Compositions and methods for inhibiting arginase activity

ABSTRACT

Compositions and methods for inhibiting arginase activity, including arginase activity in a mammal, are provided. Methods of making the compositions of the invention are also provided as are methods of using the compositions therapeutically. The compositions described herein are useful for alleviating or inhibiting a variety of arinase- and NO synthase-related disorders, including heart diseae, gastrointestinal motility disorders, and penile erectile dysfunction in humans.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of international patentapplication PCT/US98/21430, filed Oct. 9, 1998. This application is alsoentitled to priority pursuant to 35 U.S.C. §119(e) to U.S. provisionalpatent application No. 60/061,607, which was filed on Oct. 10, 1997.

STATEMENT REGARDING FEDERALLY SUPPORTED RESEARCH OR DEVELOPMENT

This research was supported in part by U.S. Government funds (U.S.National Institutes of Health grants number GM45614 and DK44841), andthe U.S. Government may therefore have certain rights in the invention.

BACKGROUND OF THE INVENTION

The invention relates generally to enzyme inhibitors, more particularlyto inhibitors of the enzyme designated arginase.

Each individual excretes roughly ten kilograms of urea per year, as aresult of the hydrolysis of arginine in the final cytosolic step of theurea cycle (Krebs et al., 1932, Hoppe-Seyler's Z. Physiol. Chem.210:33-66). The activity of the liver enzyme, arginase, permits disposalof nitrogenous wastes which result from protein catabolism (Herzfeld etal., 1976, Biochem. J. 153:469-478). In tissues which lack a completecomplement of the enzymes which catalyze the reactions of the ureacycle, arginase regulates cellular concentrations of arginine andornithine, which are used for biosynthetic reactions (Yip et al., 1972,Biochem. J. 127:893-899). Arginine is used, by way of example, in thesynthesis of nitric oxide. In macrophages, arginase activity isreciprocally coordinated with the activity of the enzyme, nitric oxidesynthase. Reciprocal coordination of the activities of arginage andnitric oxide (NO) gynthage modulates NO-dependent cytotoxicity(Corraliza et al., 1995, Biochem. Biophys. Res. Commun. 206:667-673;Daghigh et al., 1994, Biochem. Biophys. Res. Commun. 202:174-180;Chénais et al., 1993, Biochem. Biophys. Res. Commun. 196:1558-1565;Klatt et al., 1993, J. Biol. Chem. 268:14781-14787; Keller et al., 1991,Cell. Immunol. 134:249-256; Albina et al., 1995, J. Immunol.155:4391-4396).

Synthesis and evaluation of non-reactive arginine analogs for use asenzyme inhibitors or receptor antagonists is a rapidly growing area ofmedicinal chemical research (Griffith et al., 1995, Annu. Rev. Physiol.57:707-736; Gross et al., 1990, Biochem. Biophys. Res. Commun.170:96-103; Hibbs et al., 1987, J. Immunol. 138:550-565; Lambert et al.,1991, Life Sci. 49:69-79; Olken et al., 1992, J. Med. Chem.35:1137-1144; Feldman et al., 1993, J. Med. Chem. 36:491-496; Narayananet al., 1994, FASEB J. 8:A360; Narayanan et al., 1994, J. Med. Chem.37:885-887; Moore et al., 1994, J. Med. Chem. 37:3886-3888; Moynihan etal., 1994, J. Chem. Soc. Perkin Trans.769-771; Robertson et al., 1995,J. Bioorganic Chem. 23:144-151).

To date, the X-ray crystal structure of one of the enzymes of mammalianarginine catabolism, namely rat liver arginase, is available (Kanyo etal., 1996, Nature 383:554-557). Rat liver arginase is a trimericmetalloenzyme which contains a bi-nuclear manganese cluster in theactive site of each subunit. This bi-nuclear cluster is required formaximal catalytic activity (Reczkowski et al., 1992, J. Am. Chem. Soc.114:10992-10994).

As noted herein, arginase catalyzes divalent cation-dependent hydrolysisof L-arginine to form L-ornithine and urea. The enzyme is currentlyknown to serve three important functions: production of urea, productionof ornithine, and regulation of substrate arginine levels for nitricoxide synthase (Jenkinson et al., 1996, Comp. Biochem. Physiol.114B:107-132; Kanyo et al., 1996, Nature 383:554-557; Christianson,1997, Prog. Biophys. Molec. biol. 67:217-252). Urea production providesa mechanism to excrete nitrogen in the form of a highly soluble,non-toxic compound, thus avoiding the potentially dangerous consequencesof high ammonia levels. L-ornithine is a precursor for the biosynthesisof polyamines, spermine, and spermidine, which have important roles incell proliferation and differentiation. Finally, arginase modulatesproduction of nitric oxide by regulating the levels of arginine presentwithin tissues.

Since both NO synthase and arginase compete for the same substrate, thepossibility of reciprocal regulation of both arginine metabolic pathwayshas recently been explored (Modelell et al., 1995, Eur. J. Immunol.25:1101-1104; Wang et al., 1995, Biochem. Biophys. Res. Commun.210:1009-1016). Furthermore, N^(ω)-hydroxy-L-arginine (L-HO-Arg), anintermediate in the NO synthase reaction (Pufahl et al., 1992,Biochemistry 31:6822-6828; Klau et al, 1993, J. Biol. Chem.268:14781-14787; Furchgom, 1995, Annu. Rev. Pharmacol. Toxicol.,35:1-27; Yamaguchi et al., 1992, Fur. J. Biochem., 204:547-552; Pufahlet al., 1995, Biochemisty 34:1930-1941), is an endogenous arginaseinhibitor (Chenais et al., 1993, Biochem. Biophys. Res. Commun.,196:1558-1565; Buga et al., 1996, Am. J. Physiol. Heart Circ. Physiol.271:H1988-H1998 Daghigh et al., 1994, Biochem. Biophys. Res. Commun,202;174-180; Boucher et al., 1994, Biochem. Biophys. Res. Commun.203:1614-1621). The phenomenon of reciprocal regulation between arginaseand NO synthase has only recently been examined (Chakder and Rattan,1997, J. Pharmacol. Exp. Ther. 282:378-384; Langle et al., 1997,Transplantation 63:1225-1233; Langle et al., 1995, Transplantation59:1542-1549). In the internal anal sphincter (IAS), it was shown thatexogenous administration of arginase attenuates NO synthase-mediatednon-adrenergic and non-cholinergic (NANC) nerve-mediated relaxation(Chakder and Rattan, 1997, J. Pharmacol. Exp. Ther. 282:378-384).

An excess of arginase has recently been associated with a number ofpathological conditions that include gastric cancer (Wu et al., 1992,Life Sci. 51:1355-1361; Leu and Wang, 1992, Cancer 70:733-736; Straus etal., 1992, Clin. Chim. Acta 210:5-12; Ikemoto et al, 1993, Clin. Chem.39:794-799; Wu et al., 1994, Dig. Dis. Sci. 39:1107-1112), certain formsof liver injury (Ikemoto et al., 1993, Clin. Chem. 39:794-799), andpulmonary hypertension following the orthotopic liver transplantation(Langle et al., 1997, Transplantation 63:1225-1233; Langle et al., 1995,Transplantation 59:1542-1549). Furthermore, high levels of arginase cancause impairment in NANC-mediated relaxation of the IAS (Chakder andRattan, 1997, J. Pharmacol. Esp. Ther. 282:378-384). Previous studieshave demonstrated that arginase pre-treatment causes significantsuppression of the NANC nerve-mediated relaxation of the IAS (Chakderand Rattan 1997, J. Pharmacol. Exp. Ther. 282:378-384) that is mediatedprimarily via the L-arginine-NO synthase pathway (Rattan and Chakder,1992, Am. J. Physiol. Gastrointest. Liver Physiol. 262: G107-G112;Rattan and Chakder, 1992, Gastroenterology 103:43-50). Impairment inNANC relaxation by excess arginase may be related to L-argininedepletion (Wang et al., 1995, Eur. J. Immunol. 25:1101-1104).Furthermore, suppressed relaxation could be restored by the arginaseinhibitor L-HO-Arg. It is possible, therefore, that patients withcertain conditions associated with an increase in arginase activity maystand to benefit from treatment with arginase inhibitors. However, anarginase inhibitor such as L-OH-Arg can not be selective since it alsoserves as a NO synthase substrate (Pufahl et al., 1992, Biochemistry31:6822-6828; Furchgott, 1995, Annu. Rev. Pharmacol. Toxicol. 25:1-27;Pufahl et al, 1995, Biochemistry 34:1930-1941; Chemais et al., 1993,Biochem. Biophys. Res. Commun. 196:1558-1565; Boucher et al., 1994,Biochem. Biophys. Res. Commun. 203:1614-1621; Griffith and Stuehr, 1995,Annu. Rev. Physiol. 57:707-736). Because of this, the exact role ofarginase in pathophysiology and the potential therapeutic actions ofarginase inhibitors remains undetermined.

Erectile dysfunction afflicts one-half of the male population over theage of forty. This malady often results from defects in the complexcascade of enzyme-catalyzed reactions governing blood flow into and outof the corpus cavemosum, a chamber of muscular, spongy tissue thatbecomes engorged with blood in the erect penis. Defects that compromisecavemosal blood flow often occur as secondary complications related toother health conditions, such as heart disease, hypertension, diabetes,use of certain medications, and the like.

A need remains for inhibitors of arginase activity, which are useful fortreating diseases or disorders characterized either by abnormally higharginase activity in a tissue of a mammal or by abnormally low nitricoxide synthase activity in a tissue of the mammal.

SUMMARY OF THE INVENTION

The invention include a composition comprising an arginase inhibitorhaving the structure

HOOC—CH(NH₂)—X¹—X²—X³—X⁴—B(OH)₂

wherein each of X¹, X², X³, and X⁴ is selected from the group consistingof —(CH₂—)—, —S—, —O—, —(NH)—, and —(N-alkyl)—, except X² is not —S—wheneach of X¹, X³, and X⁴ is —(CH₂)—. In a preferred embodiment, theinhibitor is 2(S)-amino-6-boronohexanoic acid (ABHA), which has thestructure

HOOC—CH(NH₂)—CH₂—CH₂—CH₂—CH₂—CH₂—B(OH)₂.

The composition can further comprise a pharmaceutically acceptablecarrier.

Also included in the invention is a pharmaceutical compositioncomprising a pharmaceutical acceptable carrier and an arginase inhibitorhaving the structure

HOOC—CH(NH₂)—X¹—X²—X³—X⁴—B(OH)₂

wherein each of X¹, X², X³, and X⁴ is selected from the group consistingof —(CH₂)—, —S—, —O—, —(NH)—, and —(N-alkyl)—. For example, thestructure can be one of

HOOC—CH(NH₂)—CH₂—CH₂—CH₂—CH₂—B(OH)₂

and

HOOC—CH(NH₂)—CH₂—S—CH₂—CH₂—B(OH)₂.

The invention further includes a method of inhibiting arginase. Thismethod comprises contacting the arginage with a compound having thestructure

HOOC—CH(NH₂)—X¹—X²—X³—X⁴—B(OH)₂.

wherein each of X¹, X², X³, and X⁴ is selected from the group consistingof —(CH₂)—, —S—, —O—, —(NH)—, and —(N-alkyl)—. The arginase can, forexample, be a yeast arginase or a mammalian arginase. When the arginaseis mammalian arginase, the arginase is human arginase such as a humantype II arginase (e,g. human penile arginase).

Also included in the invention is a method of inhibiting arginase in amammal. This method comprises administering to the mammal (e.g. a human)a composition comprising a pharmaceutically acceptable carrier and anarginase inhibitor having the structure

HOOC—CH(NH₂)—X¹—X²—X³—X⁴—B(OH)₂

wherein each of X¹, X², X³, and X⁴ is selected from the group consistingof —(CH₂)—, —S—, —O—, —(NH)—, and —(N-alkyl)—. The structure can, forexample, be either of

HOOC—CH(NH₂)—CH₂—CH₂—CH₂—CH₂—B(OH)₂

and

HOOC—CH(NH₂)—CH₂—S—CH₂—CH₂—B(OH)₂.

When the mammal is a human, the human can be one who comprises either atissue which exhibits an abnormally high level of arginase activity or atissue which exhibits an abnormally low level of nitric oxide synthaseactivity.

Also included in the invention is a method of treating anarginase-related disorder in a human. This method comprisesadministering to the human a composition comprising a pharmaceuticallyacceptable carrier and an arginase inhibitor having the structure

 HOOC—CH(NH₂)—X¹—X²—X³—X⁴—B(OH)₂

wherein each of X¹, X², X³, and X⁴ is selected from the group consistingof —(CH₂)—, —S—, —O—, —(NH)—, and —(N-alkyl)—. The disorder can, forexample, be one selected from the group consisting of a disorderassociated with an abnormally low level of nitric oxide synthaseactivity in a tissue of the human and a disorder associated with anabnormally high level of arginine activity in a tissue of the human.Examples of such disorders include heart disease, systemic hypertension,pulmonary hypertension, erectile dysfunction, autoimmuneencephalomyelitis, chronic renal failure, gastrointestinal motilitydisorders, gastric cancers, reduced (or insufficient) hepatic bloodflow, and cerebral vasospasm.

The invention further includes a method of relaxing smooth muscle in amammal. This method comprises administering to the mammal a compositioncomprising a pharmaceutically acceptable carrier and an arginaseinhibitor having the structure

HOOC—CH(NH₂)—X¹—X²—X³—X⁴—B(OH)₂

wherein each of X¹, X², X³, and X⁴ is selected from the group consistingof —(CH₂)—, —S—, —O—, —(NH)—, and —(N-alkyl)—. The smooth muscle whichis relaxed according to this method can be one of a gastrointestinalsmooth muscle, anal sphincter smooth muscle, esophageal sphinctermuscle, corpus cavernosum, sphincter of Oddi, arterial smooth muscle,heart smooth muscle, pulmonary smooth muscle, kidney smooth muscle,uterine smooth muscle, vaginal smooth muscle, cervical smooth muscle,placental smooth muscle, and ocular smooth muscle.

In addition, the invention includes a method of making a compound havingthe structure

HOOC—CH(NH₂)—CH₂—CH₂—CH₂—CH₂—B(OH)₂.

This method comprises contacting a molecule of the tert-butyl ester of2(S)-N-(tert-butyloxycarbonyl)-6-[(1S,2S,3R,5S)-(+)-pinanedioxaboranyl]-hexanoicacid in an organic solvent (e.g. CH₂Cl₂) with BCl₃.

Also included in the invention is a method of making a compound havingthe structure

HOOC—CH(NH₂)—CH₂—CH₂—CH₂—CH₂—B(OH)₂.

This method comprises the steps of

(a) mixing a solution of the tert-butyl ester of2(S)-N-(tert-butyloxycarbonyl)-glutamic acid in tetrahydrofuran withtriethylamine and ethyl chloroformate to produce a first mixture,removing the resulting trimethylammonium hydrochloride salt byfiltration, and treating the remaining mixture with an aqueous solutionof sodium borohydride to provide a first compound, wherein the firstcompound is the tert-butyl ester of2(S)-N-(tert-butyloxycarbonyl)-5-hydroxypontanoic acid;

(b) subjecting the first compound to Swern oxidation to produce a secondcompound;

(c) subjecting the second compound to a Wittig reaction in the presenceof triphenylphosphonium methylide to produce a third compound;

(d) mixing a solution of BH₃ with the third compound in the presence oftetrahydrofuran to produce a second mixture,

(e) adding (1S,2S,3R,5S)-(+)-pinanediol to the second mixture to producea fourth compound, wherein the fourth compound is the tert-butyl esterof2(S)-N-(tert-butyloxycarbonyl)-6-[(1S,2S,3R,5S)-(+)-pinanedioxaboranyl]-hexanoicacid; and

(f) mixing the fourth compound with BCl₃ in the presence of CH₂Cl₂ toproduce the compound.

Also included in the invention is a method of identifying an arginaseinhibitor antagonist, the method comprising the steps of

(a) inducing relaxation of a muscle in vitro;

(b) reversing the relaxation by contacting the muscle with arginase;

(c) adding an arginase inhibitor to the muscle so reversed to renewrelaxation of the muscle in the presence or absence of a test compound;and

(d) measuring the level of renewed relaxation of the muscle, wherein alower level of renewed relaxation of the muscle in the presence of thetest compound, compared with the level of renewed relaxation of themuscle in the absence of the test compound, is an indication that thetest compound is an arginase inhibitor antagonist.

In another aspect, the invention relates to a method of alleviatingerectile dysfunction in a human. In this method, a pharmaceuticalcomposition is administered to the human, the composition comprising anarginase inhibitor having the structure

HOOC—CH(NH₂)—X¹—X²—X³—X⁴—B(OH)₂

wherein each of X¹, X², X³, and X⁴ is selected from the group consistingof —(CH₂)—, —S—, —O—, —(NH)—, and —(N-alkyl)—. Preferably, the arginaseinhibitor is ABHA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram which illustrates the topology of an arginasemonomer. Relative locations of metal ligands are indicated by solidcircles.

FIG. 2 is an image depicting a ribbon plot of an arginase trimer. TheMn²⁺—Mn²⁺ cluster in the active site of each monomer is represented by apair of spheres.

FIG. 3, comprising FIGS. 3A and 3B, is a pair of images which depictomit maps of the bi-nuclear manganese cluster of arginase. The maps werecalculated using Fourier coefficients |F₀|-|F_(c)| and phases derivedfrom the final model, less the atoms of metal ligands or metal ions Mn²⁺_(A) and Mn_(B) ²⁺. In FIG. 3A, the hydrogen bond between Oδ2 of Mn²⁺_(A) ligand Asp 128 and bridging solvent, which is represented by asphere, is indicated by a dashed line. In FIG. 3B, the manganesecoordination interactions are depicted, with coordination bond lengthsindicated in angstroms.

FIG. 4 is an image which depicts a model of arginine binding to theactive site of arginase. A salt link between Glu-277 and the substrateguanidinium group can orient the substrate for nucleophilic attack bythe metal-bridging solvent molecule. Salt links are indicated by dottedlines. His-141 can serve as a catalytic proton shuttle.

FIG. 5 is an image which illustrates a proposed mechanism ofarginase-catalyzed arginine hydrolysis by a metal-activated solventmolecule complexed with the Mn²⁺—Mn²⁺ cluster in the active site ofarginase. The alpha-amino and alpha-carboxylate groups of the argininemolecule are omitted for clarity.

FIG. 6 is an image which illustrates the synthetic scheme describedherein for the production of ABHA.

FIG. 7 is an image which depicts an ORTEP representation of themolecular structure of ABHA, using 30% probability thermal ellipsoids.

FIG. 8, comprising FIGS. 8A and 8B, is a pair of images which illustratethe similarity between the interaction of arginase with the proposedtetrahedral intermediate of arginase-catalyzed arginine hydrolysis,depicted in FIG. 8A, and the interaction of arginase with the proposedthe proposed tetrahedral conformation of hydrated ABHA, depicted in FIG.8B.

FIG. 9 is an image which depicts an omit electron density map of thearginine-ornithine-borate complex, contoured at 3σ. Refined atomiccoordinates are superimposed. One oxygen atom of the tetrahedral borateanion bridges the bi-nuclear manganese cluster. The precise conformationof ornithine (about 5 angstroms away from borate) is ambiguous, due tothe low resolution of the electron density map.

FIG. 10 is a diagram of Scheme 1 depicting the chemical structures ofcompounds 7 (ABHA), 12, 15 (BEC), and 16.

FIG. 11A is a scheme illustrating the reciprocal coordination of NOsynthase and arginase activities.

FIG. 11B is a scheme of the arginase mechanism proposed by Kanyo et al.(1996, Nature 383:554-557).

FIG. 12 is a graph depicting the Eadie-Hofstee plot for the newchromogenic substrate, 1-nitro-3-guanidinobenzene (compound 18), wherev_(o) is observed velocity and (S) is substrate concentration.

FIG. 13, comprising FIGS. 13A, 13B, 13C, and 13D is a quartet of graphsdepicting plots of v_(o)/v as a function of (I) which indicatereversible inhibition only if linear. Only the trihydroxysilane,S-(2-trihydroxysilylethyl)-L-cysteine, yields a linear plot (FIG. 13A).The boronic acid-based arginine analogs 2(S)-amino-6-boronohexanoic acid(FIG. 13B), S-(2-boronoethyl)-L-cysteine (FIG. 13C), and2(S)-amino-5-boronopentanoic acid (FIG. 13D) did not yield linear plots,indicative of irreversible inhibition. However, dialysis experimentswith 2(S)-amino-6-borono-hexanoic acid indicated that it was areversible inhibitor.

FIG. 14 comprising FIGS. 14A, 14B, and 14C is a trio of graphs depictingplots of v/v_(o) versus (I)/(E_(o)) for all the boronic acid inhibitors.Plots of v/v_(o) versus (I)/(E_(o)) typically are linear for inhibitorsthat behave as inactivators. Extrapolation of linear v/v_(o) versus(I)/(E_(o)) plots to the (I)/(E_(o)) intercept gives the turnover numberfor the inactivator. However, the (I)/(E_(o)) intercept from vlv_(o)versus (I)/(E_(o)) plots gave rise to a constant, designated the trivialname pseudo-turnover number, tn_(pseudo). Smaller tn_(pseudo) valuesindicated better inhibition. Within experimental error, boronic acidinhibitors 2(S)-amino-6-borono-hexanoic acid andS-(2-boronoethyl)-L-cysteine were essentially equipotent (FIG. 14A) and(FIG. 14B), respectively). The tn_(pseudo) for boronic acid2(S)-amino-5-boronopentanoic acid (FIG. 14C) was two orders of magnitudehigher than the tn_(pseudo) values for the other two boronates. Thefirst two boronates were about 400-fold more potent than2(S)-amino-5-boronopentanoic acid.

FIG. 15 comprising FIGS. 15A and 15B, is two graphs depicting thetitration of arginase by ABHA (“7”) in a solution comprising 100micromolar MnCl₂ and 50 millimolar bicine (pH 8.5) at 25° C. FIG. 15Acontains the raw data obtained by titration of 0.0358 millimolararginase with 30×2.5 microliters injections of 1.5 millimolar ABHA. InFIG. 15B, the area under each peak was integrated and plotted against[ABHA]/[arginase]. The solid line represents the best fit of theexperimental data using non-linear least squares fitting, indicating astoichiometry (n) of 1.07 moles of bound ABHA per mole of arginasemonomer, an association constant (K_(a)) of 8.89×10⁶ inverse molar, andan enthalpy change (ΔH) of −12.97 kilocalories per mole.

FIG. 16, comprising FIGS. 16A and 16B, is a pair graphs depicting thetitration of arginase by compound 15 in a solution comprising 100micromolar MnCl₂ and 50 millimolar bicine (pH 8.5) at 25° C. FIG. 16Adepicts the raw data obtained by titration of 0.0358 millimolar arginasewith 40×2.5 microliters injections of 1.5 millimolar compound 15. InFIG. 16B the area under each peak was integrated and plotted against[15]/[arginase]. The solid line represents the best fit of theexperimental data using non-linear least squares fitting, indicating astoichiometry (n) of 0.964 moles of bound 15 per mole of arginase, anassociation constant (K_(a)) of 4.50×10⁵ inverse molar, and an enthalpychange (ΔH) of −12.75 kilocalories per mole.

FIG. 17 depicts the proposed binding mode for arginase inhibitors 7, 12,and 15. The metal-bridging hydroxide ion of the native arginase likelyattacks the trigonal planar boronic acid to form the tetrahedralboronate anion.

FIG. 18 is a graph depicting the effect of exogenous arginase, beforeand after application of the arginase inhibitor N^(ω)-hydroxy-L-Arginine(L-HO-Arg), on the IAS relaxation by different frequencies of electricalfield stimulation (EFS). Note the significant suppression of the IASrelaxation by arginase alone and its reversal by L-HO-Arg.

FIG. 19 is a graph depicting the effect of exogenous arginase treatment,before and after application of the arginase inhibitor2(S)-Amino-6-boronohexanoic Acid (ABHA), on IAS smooth muscle relaxationby different frequencies of EFS. Note the significant suppression of theIAS relaxation by treatment with arginase alone, and its reversal byABHA.

FIG. 20 is a graph depicting the percent maximal decrease in IAS tensionby EFS before and after treatment of the tissue with the NO synthaseinhibitor L-NNA or L-NNA in combination with selected concentrations ofL-HO-Arg. L-NNA caused a marked attenuation of EFS-induced IASrelaxation (p<0.05; n=5). L-NNA-attenuated IAS relaxation was reversedby L-HO-Arg in a concentration-dependent manner. L-HO-Arg (3×10⁻⁴ molar)caused complete reversal of suppressed IAS relaxation.

FIG. 21 is a graph depicting the percent maximal decrease in IAS tensionproduced by EFS, before and after application of the NO synthaseinhibitor L-NNA or L-NNA in combination with selected concentrations ofL-arginine. Note that L-arginine caused a significant andconcentration-dependent reversal of suppressed IAS relaxation and thatthis effect was somewhat similar to that of L-HO-Arg (p<0.05; n=7).

FIG. 22 is a graph depicting the percent maximal decrease in IAS tensionproduced by EFS before and after application of the NO synthaseinhibitor L-NNA or L-NNA in combination with selected concentrations ofABHA. Note that unlike L-HO-Arg, ABHA had no effect on theL-NNA-suppressed IAS relaxation (p<0.05; n=4).

FIG. 23 is a graph depicting the influence of L-HO-Arg on NANCnerve-mediated IAS relaxation produced by EFS. Note that L-HO-Arg causeda significant augmentation of the EFS-induced relaxation of IAS in aconcentration-dependent manner and this was evident only in the lowerfrequencies of EFS (p<0.05).

FIG. 24 is a graph depicting the influence of ABHA on NANCnerve-mediated IAS relaxation. Note that ABHA caused a significantaugmentation of IAS smooth muscle relaxation caused by the lowerfrequencies of EFS, in a concentration-dependent manner (p<0.05).

FIG. 25 is a graph depicting basal arginase activity in tissuehomogenates of liver, internal anal sphincter (IAS), rectum, and brain.Basal arginase activity for the non-hepatic tissues was found to beabout 10⁻³ of that in liver tissue. Interestingly, among the non-hepatictissues, basal arginase activity in IAS smooth muscle was found to benearly four-fold higher than in either the adjoining region of therectum or the brain.

FIG. 26 is a graph depicting the effect of N^(ω)-hydroxy-L-arginine onarginase activity in hepatic and non-hepatic tissues.N^(ω)-hydroxy-L-arginine was found to be about 10 times more potent ininhibiting liver arginase activity than arginase activities innon-hepatic tissues.

FIG. 27 is a graph depicting a comparison of the effect of ABHA onhepatic versus non-hepatic tissues (IAS, rectum, and brain). In contrastto the effects seen with N^(ω)-hydroxy L-arginine, ABHA was a morepotent inhibitor of non-hepatic than hepatic arginase activities.

FIG. 28, comprising FIGS. 28A-28D, depicts the influence of the NOsynthase inhibitor L-NNA, N^(ω)-hydroxy-L-arginine (L-HO-Arg), and thecombination of L-NNA and L-HO-Arg on liver arginase activity (FIG. 28A),IAS arginase activity (FIG. 28B), rectum arginase activity (FIG. 28C),and brain arginase activity (FIG. 28D). These data illustrate that L-NNAhad no significant effect on either basal arginase activity orL-HO-Arg-attenuated arginase activity in the tissues examined. The datasuggest that the differential inhibitory effects of L-HO-Arg in thesetissues do not result from variable interactions of this NO synthasesubstrate with endogenous NO synthase.

FIG. 29, comprising FIGS. 29A, 29B, and 29C is a trio of diagrams whichdepict the structure and arrangement of the arginase-ABHA complex. FIG.29A is an Omit electron density map, generated using BOBSCRIPT andRaster3D software (Esnouf, 1997, J. Mol. Graphics 15:132-134; Merritt etal., 1997, Methods Enzymol. 277:505-524) of ABHA in the arginase activesite averaged over the two monomers in the asymmetric unit and averagedover the two twin domains A and B, as described herein. The map in FIG.29A is contoured at 7.7σ and selective active site residues areindicated. Atoms are color-coded as follows: C (yellow), O (red), N(blue, and B (pale green). Water molecules in FIG. 29A are shown as redspheres. FIG. 29B is a summary of arginase-ABNA interactions. FIG. 29Cis a diagram which illustrates stabilization of the tetrahedralintermediate (and flanking transition states) in the arginase mechanism,based on the binding mode of ABHA.

FIG. 30, comprising FIGS. 30A, 30B, and 30C, illustrates the effect ofABHA on NANC nerve-mediated relaxation in penile corpus cavemosum smoothmuscle tissue. FIG. 30A illustrates representative polygraph tracing ofresponses to EFS in the absence and presence of 1 millimolar ABHA. InFIG. 30A, tissue tone is represented as grams of tension on theordinate. In the absence of electrical stimulation, ABHA caused moderaterelaxation, due to basal activity of NO synthase. FIG. 30B is a bargraph which summarizes data gathered in organ bath experiments. Allresponses in the presence of ABHA were significantly greater (p≦0.05),with the exception of the response obtained at 1 Hertz in the presenceof 0.1 millimolar ABHA. FIG. 30C is a bar graph which illustratesincrease in relaxation caused by ABHA, relative to control responses.Enhancement by ABHA was statistically significant (p≦0.05) at allconcentrations and frequencies tested.

DETAILED DESCRIPTION

The invention is based upon the discovery of compounds which inhibit theenzymatic activity of arginase. These compounds, which were notpreviously known to inhibit this enzyme (and at least some of which wereapparently not previously known to have any use), are useful for avariety of applications in medicine and research.

Compositions and methods for inhibiting the activity of arginaseincluding, but not limited to, yeast and mammalian arginase, aredescribed herein. Inhibition of mammalian arginase activity using theMoronic acid-based arginine analog 2(S)-amino-6-boronohexanoic acid(ABHA) is also described herein, as is inhibition of arginase using theboronic acid based arginine analog S-(2-boronoethyl)-L-cysteine (BEC).

The compositions described herein can be used to inhibit arginaseactivity in vitro or in vivo, for example, in a human. Thesecompositions can also be used to treat a disorder characterized eitherby abnormally high arginase activity in a tissue of a mammal or byabnormally low nitric oxide synthase activity in a tissue of the mammal,preferably a human.

The composition of the invention comprises an arginase inhibitor havingthe structure

HOOC—CH(NH₂)—X¹—X²—X³—⁴—B(OH₂

wherein each of X¹, X², X³, and X⁴ is selected from the group consistingof —(CH₂)—, —S—, —O—, —(NH)—, and —(N-alkyl)—, except X² is not —S— wheneach of X¹, X³, and X⁴ is —(CH₂)—.

In one aspect, the arginase inhibitor has the structure

HOOC—CH(NH₂)—CH₂—CH₂—CH₂—CH₂—B(OH)₂.

This compound is 2(S)-amino-6-boronohexanoic acid (ABHA). ABHA isalternatively referred to herein as compound 7. Data presented hereinconfirm that ABHA is a potent inhibitor of arginase activity. Theconformation of the hydrated form of ABHA resembles a transition stateintermediate postulated to be formed during the arginine hydrolysisreaction catalyzed by arginase. The tetrahedral structure around theboron atom of hydrated arginase (e.g. ABHA, as depicted in FIG. 8B)closely resembles the tetrahedral intermediate formed by hydroxyl ionnucleophilic attack at the guanidinium carbon of arginine.

In a preferred embodiment, the composition of the invention furthercomprises a pharmaceutically acceptable carrier, go as to render thecomposition suitable for administration to a human or another mammal.

Also included in the invention is a composition comprising apharmaceutical acceptable carrier and an arginase inhibitor having thestructure

HOOC—CH(NH₂)—X¹—X²—X³—X⁴—B(OH)₂

wherein each of X¹, X², X³, and X⁴ is selected from the group consistingof —(CH₂)—, —S—, —O—, —(NH)—, and —(N-alkyl)—.

Preferred compositions which also include a pharmaceutically acceptablecarrier, include ABHA and a compound having the structure

HOOC—CH(NH₂)—CH₂—S—CH₂—CH₂—B(OH)₂

This inhibitor is S-(2-boronoethyl)-L-cysteine (BEC). BEC is alsoreferred to herein as compound 15.

Any physiologically acceptable anion can be used as a counter-ion whenan arginase inhibitor described herein is prepared in the form of asalt. Physiologically acceptable anions are known in the art, andinclude, for example, chloride, acetate, and citrate.

The invention includes a method of inhibiting arginase, the methodcomprising contacting a composition comprising an arginase inhibitordescribed herein with an arginase enzyme, such as a yeast arginaseenzyme or a mammalian arginase enzyme. When the enzyme is a mammalianarginase, the arginase is preferably a human arginase enzyme, and morepreferably a human type II (i.e. non-hepatic type) arginase.

“Inhibition” of arginase by an arginase inhibitor means reduction in thelevel of arginase activity in the presence of the inhibitor, comparedwith the level of arginase activity in the absence of the inhibitor.

Arginase can be inhibited in yeast by contacting the yeast with thecomposition of the invention. Inhibition of arginase in yeast serves tominimize urea production during fermentation of alcoholic beverages.

In addition, the compositions of the invention are useful asanti-fungicides in agriculturally or otherwise economically importantplant life. The composition of the invention can be administered to theplant by spraying or other means well known in the art of plant biology.

Arginase activity can be inhibited in a mammal, for example, byadministering a pharmaceutical composition comprising an arginaseinhibitor described herein to the mammal.

Thus, the composition of the invention can be used to treat a disorderin a mammal, wherein the disorder is associated with expression of anabnormally high level of arginase activity in a tissue of the mammal.Because NO synthase activity is regulated in a reciprocal fashion withrespect to arginase activity in mammals, more particularly humans, thecomposition of the invention can be used to treat a disorder in amammal, wherein the disorder is associated with expression of anabnormally low level of NO synthase activity in a tissue of the mammal.Since the reciprocal interaction of arginase and NO synthase hasimplications for the function of smooth muscle as described in furtherdetail herein in Example 4, the use of the compounds described hereinfor the regulation of smooth muscle activity in an animal is alsocontemplated in the invention. Of course, a composition which comprisesan arginase inhibitor described herein can also be used to inhibitarginase in a mammal having normal levels of arginase and NO synthaseactivity, particularly where the physiological which is desired to beeffected is one which is affected by arginase or NO synthase activity,or where a disorder which is not caused by aberrant arginase or NOsynthase activity levels can nonetheless be alleviated or inhibited byinhibiting arginase activity (e.g. certain forms of erectiledysfunction).

An “abnormally high level of arginase activity” means a level ofarginase activity which exceeds the level found in normal tissue whenthe normal tissue does not exhibit an arginase related disorderphenotype.

An “abnormally low level of NO synthase activity” means a level of NOsynthase activity which is lower than that found in normal tissue whenthe normal tissue does not exhibit an NO synthase related disorderphenotype.

An increase in arginase activity has been associated with thepathophysiology of a number of conditions including impairment innon-adrenergic and non-cholinergic (NANC) nerve-mediated relaxation ofgastrointestinal smooth muscle. An arginase inhibitor can be used toalleviate such impairment by administering the inhibitor to a mammalexperiencing such impairment or a mammal which is anticipated toexperience such impairment (e.g. a human afflicted with agastrointestinal motility disorder).

Thus, the invention includes a method of enhancing smooth musclerelaxation comprising contacting the smooth muscle with an arginaseinhibitor. The smooth muscle is preferably within the body of an animaland the arginase inhibitor is preferably ABHA or BEC. The type of smoothmuscle to be relaxed includes, but is not limited to, gastrointestinalsmooth muscle, anal sphincter smooth muscle, esophageal sphinctermuscle, sphincter of Oddi, arterial smooth muscle, heart smooth muscle,pulmonary smooth muscle, kidney smooth muscle, uterine smooth muscle,vaginal smooth muscle, cervical smooth muscle, placental smooth muscle,and ocular smooth muscle. When the smooth muscle is gastrointestinalsmooth muscle, the type of gastrointestinal smooth muscle includes, butis not limited to, the internal anal sphincter muscle.

In an important embodiment, the invention relates to use of an arginaseinhibitor described herein for enhancing penile erectile function in amammal (preferably a human) or for alleviating erectile dysfunction in amammal.

NO is an important regulator of erectile function and mediates NANCneurotransmission in penile corpus cavernosum smooth muscle, leading torapid relaxation, which in turn leads to erection. NO synthase, whichcatalyzes oxidation of L-arginine to form L-citrulline and NO, is forthis reason a key enzyme in penile smooth muscle physiology.

Arginase catalyzes hydrolysis of L-arginine to form L-ornithine andurea. Arginase regulates NO synthase activity by affecting the amount ofL-arginine available for oxidation catalyzed by NO synthase activity.Thus, inhibition of arginase activity can enhance NO synthase activity,thereby enhancing NO-dependent smooth muscle relaxation in the corpuscavemosum and enhancing penile erection.

When the smooth muscle in within the body of the animal, the inventionincludes a method of alleviating (e.g. reducing the incidence orseverity) or inhibiting (e.g. reducing the likelihood of developing, orpreventing) an arginase-related disorder in an animal. In a preferredembodiment, the animal is a human.

Disorders which are associated with either an abnormally high level ofarginase activity in a tissue of a mammal or an abnormally low level ofnitric oxide synthase activity in a tissue of the mammal are known inthe art. Disorders to be treated using the compositions of the inventioninclude, but are not limited to, heart disease, systemic hypertension,pulmonary hypertension, erectile dysfunction, autoimmuneencephalomyelitis, chronic renal failure, gastrointestinal motilitydisorders, gastric cancers, reduced hepatic blood flow, and cerebralvasospasm.

To alleviate an arginase-related disorder in a mammal, an arginineinhibitor described herein is administered to a mammal afflicted withthe disorder. The inhibitor is preferably administered in combinationwith one or more pharmaceutically acceptable carriers, as described infurther detail herein. The inhibitor (preferably in combination with acarrier) can also be administered to a mammal afflicted with a disordercharacterized by aberrant NO synthase activity, or to one which exhibitsnormal (i.e. non-diseased) levels of arginase and NO synthaseactivities, but in which inhibition of arginase activity is desired. Theinvention also contemplates use of an arginase inhibitor in an in vitroarginase inhibition/smooth muscle relaxation functional assay, for thepurpose of identifying compounds which affect smooth muscle function.Compounds so identified are considered to be candidate arginaseinhibitor antagonists, in that, as described in further detail below,these compounds are identified by their ability to counteract the ABHAor BEC mediated inhibition of arginase activity. For example, there isdescribed herein in Example 4 an assay for smooth muscle activity usingthe internal anal sphincter muscle and one on the preferred arginaseinhibitors of the invention ABHA. In this assay, strips of the internalanal sphincter muscle obtained from a mammal (e.g. an adult opossum) areinduced to relax by NANC nerve-mediated relaxation using electricalfield stimulation (EFS); relaxation is reversed by contacting the musclestrips with arginase; and reversal of relaxation is accomplished bycontacting the muscle with an arginase inhibitor. To identify anarginase inhibitor antagonist, the muscle strips are then subsequentlycontacted with a test compound. The effect of the test compound onsubsequent reversal of muscle relaxation is assessed. Any significantreversal of the relaxation state of the muscle in the presence of thetest compound, compared with the relaxation state of the muscle in theabsence of the test compound, is an indication that the test compound isan arginase inhibitor antagonist.

An “arginase inhibitor antagonist” means a compound which reduces orprevents inhibition of arginase by an arginase inhibitor.

The invention encompasses preparation and use of pharmaceuticalcompositions comprising an arginase inhibitor described herein as anactive ingredient. Such a pharmaceutical composition can consist of theactive ingredient alone, in a form suitable for administration to asubject, or the pharmaceutical composition can comprise the activeingredient and one or more pharmaceutically acceptable carriers, one ormore additional ingredients, or some combination of these.Administration of one of these pharmaceutical compositions to a subjectis useful for inhibiting arginase activity and thereby treating adisease or disorder associated with arginase enzyme activity, asdescribed elsewhere in the present disclosure. The active ingredient canbe present in the pharmaceutical composition in the form of aphysiologically acceptable ester or salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which the active ingredient can be combinedand which, following the combination, can be used to administer theactive ingredient to a subject.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, which isnot deleterious to the subject to which the composition is to beadministered.

The formulations of the pharmaceutical compositions described herein canbe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs,birds including commercially relevant birds such as chickens, ducks,goose, and turkeys, fish including farm-raised fish and aquarium fish,and crustaceans such as farm-raised shellfish.

Pharmaceutical compositions that are useful in the methods of theinvention can be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,buccal, ophthalmic, or another route of administration. Othercontemplated formulations include projected nanoparticles, liposomalpreparations, resealed erythrocytes containing the active ingredient,and immunologically-based formulations.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which thee composition is to be administered. By way ofexample, the composition can comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention can further comprise one or more additionalpharmaceutically active agents. Particularly contemplated additionalagents include anti-emetics and scavengers such as cyanide and cyanatescavengers.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention can be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitablefor oral administration can be prepared, packaged, or sold in the formof a discrete solid dose unit including, but not limited to, a tablet, ahard or soft capsule, a cachet, a troche, or a lozenge, each containinga predetermined amount of the active ingredient. Other formulationssuitable for oral administration include, but are not limited to, apowdered or granular formulation, an aqueous or oily suspension, anaqueous or oily solution, or an emulsion.

As used herein, an “oily” liquid is one which comprises acarbon-containing liquid molecule and which exhibits a less polarcharacter than water.

A tablet comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets can be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets can be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface active agentsinclude, but are not limited to, sodium lauryl sulfate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Tablets can be non-coated or they can be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate can be used to coat tablets. Furtherby way of example, tablets can be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to formosmotically-controlled release tablets. Tablets can further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide pharmaceuticallyelegant and palatable preparation.

Hard capsules comprising the active ingredient can be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and can further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient can be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which can be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the inventionwhich are suitable for oral administration can be prepared, packaged,and sold either in liquid form or in the form of a dry product intendedfor reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions can be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions can further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions can further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solventscan be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention can comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention can be prepared using known methods. Such formulations canbe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations can further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, can also be included in these formulations.

A pharmaceutical composition of the invention can also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase can be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions can further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions can also containadditional ingredients including, for example, sweetening or flavoringagents.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in a formulation suitable for rectal administration. Such acomposition can be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

Suppository formulations can be made by combining the active ingredientwith a non-irritating pharmaceutically acceptable excipient which issolid at ordinary room temperature (i.e. about 20° C.) and which isliquid at the rectal temperature of the subject (i.e. about 37° C. in ahealthy human). Suitable pharmaceutically acceptable excipients include,but are not limited to, cocoa butter, polyethylene glycols, and variousglycerides. Suppository formulations can further comprise variousadditional ingredients including, but not limited to, antioxidants andpreservatives.

Retention enema preparations or solutions for rectal or colonicirrigation can be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations can be administered using, and can be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations can farther comprise various additional ingredientsincluding, but not limited to, antioxidants and preservatives.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in a formulation suitable for vaginal administration. Such acomposition can be in the form of, for example, a suppository, animpregnated or coated vaginally-insertable material such as a tampon, adouche preparation, or a solution for vaginal irrigation.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e. such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

Douche preparations or solutions for vaginal irrigation can be made bycombining the active ingredient with a pharmaceutically acceptableliquid carrier. As is well known in the art, douche preparations can beadministered using, and can be packaged within, a delivery deviceadapted to the vaginal anatomy of the subject. Douche preparations canfurther comprise various additional ingredients including, but notlimited to, antioxidants, antibiotics, antifungal agents, andpreservatives.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue, so long asthe arginase inhibitor compound is not administered systemically.Parenteral administration thus includes, but is not limited to,administration of a pharmaceutical composition by injection of thecomposition, by application of the composition through a surgicalincision, by application of the composition through a tissue-penetratingnon-surgical wound, and the like. In particular, parenteraladministration is contemplated to include, but is not limited to,subcutaneous, intraperitoneal, intramuscular, intrasternal injection,and kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations can be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations can be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations can further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e. powder or granular) form for reconstitution with asuitable vehicle (e.g. sterile pyrogen-free water) prior to parenteraladministration of the reconstituted composition.

The pharmaceutical compositions can be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution can be formulated according to the knownart, and can comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations can beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation can comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient can be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration can further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation can comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers, and preferably from about 1 toabout 6 nanometers. Such compositions are conveniently in the form ofdry powders for administration using a device comprising a dry powderreservoir to which a stream of propellant can be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the active ingredient dissolved orsuspended in a low-boiling propellant in a sealed container. Preferably,such powders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers. Morepreferably, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositionspreferably include a solid fine powder diluent such as sugar and areconveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant can constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient can constitute 0.1 to 20% (w/w) of the composition.The propellant can further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent(preferably having a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery can also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations can be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andcan conveniently be administered using any nebulization or atomizationdevice. Such formulations can further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration preferably have an averagediameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to 500 micrometers. Such a formulation is administered inthe manner in which snuff is taken i.e. by rapid inhalation through thenasal passage from a container of the powder held close to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and can further comprise one or more of theadditional ingredients described herein.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, 0.1 to 20% (w/w)active ingredient, the balance comprising an orally dissolvable ordegradable composition and, optionally, one or more of the additionalingredients described herein. Alternately, formulations suitable forbuccal administration can comprise a powder or an aerosolized oratomized solution or suspension comprising the active ingredient. Suchpowdered, aerosolized, or aerosolized formulations, when dispersed,preferably have an average particle or droplet size in the range fromabout 0.1 to about 200 nanometers, and can further comprise one or moreof the additional ingredients described herein.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in a formulation suitable for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.1-1.0% (w/w) solution or suspension of the activeingredient in an aqueous or oily liquid carrier. Such drops can furthercomprise buffering agents, salts, or one or more other of the additionalingredients described herein. Other ophthalmalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form or in a liposomal preparation.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which can beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed., 1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which isincorporated herein by reference.

Typically dosages of the arginase inhibitor which can be administered toan animal, preferably a human, range in amount from 1 microgram to about100 grams per kilogram of body weight of the animal. While the precisedosage administered will vary depending upon any number of factors,including but not limited to, the type of animal and type of disorderbeing treated, the age of the animal and the route of administration.Preferably, the dosage of the compound will vary from about 1 milligramto about 10 grams per kilogram of body weight of the animal. Morepreferably, the dosage will vary from about 10 milligrams to about 1gram per kilogram of body weight of the animal.

The compound can be administered to an animal as frequently as severaltimes daily, or it can be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even leesfrequently, such as once every several months or even once a year orless. The frequency of the dose will be readily apparent to the skilledartisan and will depend upon any number of factors, such as, but notlimited to, the type and severity of the disorder being treated, thetype and age of the animal, etc.

The invention is now described with reference to the following examples.These examples are provided for the purpose of illustration only and theinvention is not limited to these examples, but instead encompasses allvariations which are evident as a result of the teaching providedherein.

EXAMPLE 1 Structure of a Unique Bi-nuclear Manganese Cluster in Arginase

The molecular structure of and surrounding the pair of manganese atomsof the arginase monomer was determined by X-ray diffraction and othermethods. These data suggest the mechanism by which arginine hydrolysisis catalyzed by arginase and facilitate design of arginase inhibitorswhich mimic the transition state intermediates of the reaction, asdescribed in Example 2.

The materials and methods used in the experiments presented in thisExample are now described.

Crystallization and preliminary X-ray diffraction analysis of rat liverarginase has been reported (Kanyo et al., 1992, J. Mol. Biol.224:1175-1177). Arginase crystals diffract to 2.1 angstrom resolutionand belong to space group P3₂, having hexagonal unit-cell dimensions ofa=b=88.5 angstroms, and c=106.2 angstroms, and having one 105 kilodaltontrimer in the asymmetric unit. Phase determination by multipleisomorphous replacement was hindered by chronic non-isomorphism betweennative and heavy-atom derivative crystals, as well as non-isomorphismamong native crystals themselves. The c-axis length typically rangedfrom 104 angstroms to 115 angstroms (Kanyo et al., 1992, J. Mol. Biol.224:1175-1177).

Diffraction data were collected at room temperature on an R-AXIS IIcimage plate area detector. Data reduction was performed by MOSFLM(Nyborg et al., 1977, In: The Rotation Method in Crystallography, Arndtet al., eds, North-Holland, Amsterdam, 139-152) and CCP4 (CollaborativeComputational Project No. 4, 1994, Acta Crystallogr. D50:760-763) asdescribed. For phasing, initial heavy atom positions were determined indifference Patterson maps and refined with the program, PHASES (Burey etal., 1990, Am. Crystallogr. Assoc. Mtg. Prog. Abstr. 18:73). Heavy-atombinding indicated that the non-crystallographic symmetry (NCS) axis ofthe trimer was tilted about 9° away from a normal to the a-b plane. Themodel was fit into an electron-density map calculated withsolvent-flattened NCS-averaged phases at 3.0 angstrom resolution.Subsequent refinement and rebuilding of the native model was performedusing X-PLOR (Brdnger et al., 1987, Science 235:458-460) and O (Jones etal., 1991, Acta. Crystallogr. A47:110-119) algorithms, respectively.Group B factors were refined, and a bulk solvent correction was applied.In the final stages of refinement, the quality of the model was improvedby gradually releasing NCS constraints into appropriately weightedrestraints as judged by R_(free). Refinement statistics are recorded inTable 1. the final protein model has excellent stereochemistry with onlyGln-64 adopting a disallowed Φ/φ conformation. This residue is locatedin a type II′ beta-turn between strand 2 and helix B and ischaracterized by clear and unambiguous electron density.

The results of the experiments presented in this Example are nowdescribed.

Arginase is one of the very few enzymes that requires a spin-coupledMn²⁺—Mn²⁺ cluster for catalytic activity in vitro and in vivo(Reczkowski et al., 1992, J. Am. Chem. Soc. 114:10992-10994). The 2.1angstrom-resolution crystal structure of trimeric rat liver arginasereveals that this unique metal cluster resides at the bottom of anactive-site cleft that is about 15 angstroms deep (Kanyo et al., 1992,J. Mol. Biol. 224:1175-1177). Analysis of the crystal structure ofarginase indicates that arginine hydrolysis involves a metal-activatedsolvent molecule which symmetrically bridges the two Mn²⁺ ions.

TABLE 1 Data collection and refinement statistics. CompletenessR_(merge) ^(a) Overall Resolution Reflections (%) overall/outeroverall/outer Number Phasing Figure Data Set (angstroms) measured/uniqueshell shell R_(iso) ^(b) of Sites power^(c) of merit Native 2.1147,454/46,162  91.6/58.3 0.058/0.365 — — — — Thimerosal 3.039,030/17,363 93.7/96.5 0.063/0.226 0.199 6 1.51 — YbCl₃ 3.029,644/15,182 81.9/86.0 0.042/0.146 0.143 6 1.46 — HgAc₂ 3.026,788/16,202 87.2/91.6 0.089/0.272 0.127 6 1.78 — 0.496 No. RefinementMn²⁺ Solvent Resolution Reflections R.m.s. Deviations Protein Atoms ionsmolecules (angstroms) work/free R/R_(free) ^(d) Bonds Angles DihedralsImproper Native 7,167 6 233 2.1 47,913/ 0.179/ 0.011 1.6° 24.7° 1.6°2,502 0.229 Å ^(a)R_(merge) = Σ|I_(i) − <I_(t)>|/Σ|<I_(i)>|, where I_(i)is the intensity measurement for reflection i, and <I_(i)> is the meanintensity calculated for reflection i from replicate data. ^(b)R_(iso) =Σ||F_(PH)| − |F_(P)||/Σ|F_(P)|, where F_(PH) and F_(P) are thederivative and native structure factors, respectively. ^(c)Phasing power= <F_(h)>/E, where <F_(h)> is the root-mean-square heavy atom structurefactor and E is the residual lack of closure error. ^(d)R = ||F_(o)| −|F_(c)||/Σ|F_(o)|, where R and R_(free) are calculated using the workingand free reflections sets, respectively. The free reflections,representing 5% of the total, were held aside throughout refinement.

The overall folding pattern of the arginase monomer indicates that theprotein belongs to the alpha/beta protein class. The arginase monomerhas a globular structure, having approximate dimensions of 40×50×50angstroms. One side of the active-site cleft is partially defined by thecentral eight-stranded beta-sheet, and the metal binding site is locatedon one edge of the beta-sheet. Metal ligands are located immediatelyadjacent to helix C, to strand 4, and to strand 7, as depicted in FIG.1. The arginase trimer, depicted in FIG. 2, has a relative molecularmass of about 105,000 (M_(r)=105 kilodaltons) and excludes about 2,080square angstroms of monomer surface area from solvent at eachmonomer-monomer interface. A majority of inter-monomer contact ismediated by a novel ‘S’-shaped oligomerization motif at the carboxylterminus, as depicted in FIGS. 1 and 2, and the conformation of thissegment is stabilized by numerous inter-monomer van der Waalsinteractions, hydrogen bonds, and salt links. The image in FIG. 2 wasgenerated using MOLSCRIPT (Kraulis et al., 1991, J. Appl. Crystallogr.24:946-950) and Raster 3D (Bacon et al., 1988, J. Mol. Graph. 6:219-200;Merrit et al., 1994, Acta Crystallogr. D50:869-873).

The bi-nuclear manganese cluster is located at the base of anapproximately 15 angstrom-deep active site cleft in each monomer, asdepicted in FIG. 4. The metal ion that is more deeply situated in theactive site cleft is designated Mn²⁺ _(B) and is coordinated by His-101(Nδ), Asp-124 (Oδ1), Asp-128 (Oδ1), Asp-232 (Oδ1) and a solventmolecule. The coordination of Mn²⁺ _(A) by arginase has square pyramidalgeometry. A solvent molecule bridges Mn²⁺ _(A) and Mn²⁺ _(B) and alsodonates a hydrogen bond to Asp-128, the Oδ2-O separation distance beingabout 2.8 angstroms. The second metal ion is designated Mn²⁺ _(B) and iscoordinated by His-126 (Nδ), Asp-124 (Oδ2), Asp-232 (Oδ1), Asp-234(bi-dentate Oδ1 and Oδ2). The coordination of Mn²⁺ _(B) by arginase andthe bridging solvent molecule has an octahedral geometry which isdistorted, owing to the bi-dentate coordination of Asp-234. Theseparation between Mn²⁺ _(A) and Mn²⁺ _(B) is about 3.3 angstroms. Allmetal ligands except Asp-128 make hydrogen-bond interactions with otherprotein residues, and these interactions contribute to the stability ofthe metal binding site (Christianson et al., 1989, J. Am. Chem. Soc.111:6412-6419).

Three different types of metal bridging ligands facilitate the observedspin coupling between Mn²⁺ _(A) and Mn²⁺ _(B) (Reczkowski et al., 1992,J. Am. Chem. Soc. 114:10992-10994). The carboxylate side chain ofAsp-124 is a syn-syn bi-dentate bridging ligand, with Oδ1 coordinated toMn²⁺ _(A) and Oδ2 coordinated to Mn²⁺ _(B) (Rardin et al., 1991, New J.Chem. 15:417-430). The carboxylate side chain of Asp-232 is amonodentate bridging ligand, with Oδ1 coordinated to both Mn²⁺ _(A) andMn²⁺ _(B) with anti- and syn-coordination stereochemistry, respectively(Rardin et al., 1991, New J. Chem. 15:417-430). The Oδ2 oxygen ofAsp-232 is about 3.8 angstroms away from Mn_(B) ²⁺. The solvent moleculebridges both manganese ions symmetrically, with the separation distanceof the O atom of the solvent from both of Mn²⁺ _(A) and Mn²⁺ _(B) beingabout 2.4 angstroms.

The arginase structure is the first atomic resolution structuredetermined for a functional metalloenzyme that has a specific catalyticand physiological requirement for two Mn²⁺ ions. Arginase does not usetransition metals promiscuously for catalysis, unlike othermetalloenzymes, in which Mn²⁺ and, for instance, Mg²⁺ or other metalions are interchangeable, albeit with some effect on catalytic activity.The identity and oxidation state of the manganese cluster of arginasehas been conclusively established by electron paramagnetic resonancespectroscopy (Reczkowski et al., 1992, J. Am. Chem. Soc.114:10992-10994). The catalytic metal requirement of arginase is rootedin the preferred geometry of manganese coordination, which properlyorients the metal-bridging solvent molecule, such that it arginasehydrolysis is catalyzed. Because the metal-bridging solvent moleculemust satisfy the coordination preferences of two manganese ionssimultaneously, the position of the solvent molecule, and therefore itsoptimal catalytic activity, is highly sensitive to substitution of oneor both Mn²⁺ ions with a different metal ion. Coordination of thesolvent molecule to two metal ions, rather than one, enhances thedependence of optimal catalytic activity on proper metal composition ofarginase.

Only two other polar residues are found in the immediate vicinity of theactive site of arginase, namely Glu-277 and His-141. Glu-277 is locateddeep in the active-site cleft, about 4.5 angstroms away from Mn²⁺ _(A)roughly 20° rotation about χ₁ of this side chain yields an ideal saltlink with the substrate guanidinium group. Moreover, this salt linkpositions the electrophilic guanidinium carbon of the substrate directlyover the metal-bridging solvent molecule, which is likely to be anucleophilic hydroxide ion in the active catalyst, as depicted in FIGS.4 and 5. It is unlikely that the de-protonated substrate guanidiniumgroup binds directly to the metal(s) because of its high pK_(a) value,namely 13.5. Furthermore, site-directed mutagenesis studies indicatethat substrate K_(m) values are not perturbed by variations in the metalcluster, including metal depletion. This indicates that asubstrate-metal interaction does not occur in the Michaelis complex(Cavalli et al., 1994, Biochemistry 33:10652-10657).

The side chain of His-141 is located about midway between the top andthe bottom of the active-site cleft. When asparagine is substituted inplace of histidine at amino acid position 141 of arginase (i.e.His-141→Asn arginase), the enzyme retains roughly ten percent of itswild type activity (Cavalli et al., 1994, Biochemistry 33:10652-10657).Because its location is only about 4.2 angstroms from the metal-bridgingsolvent molecule, it is possible that His-141 acts a proton shuttle inarginase catalysis, mediating proton transfer to and from bulk solvent.Direct proton transfer with bulk solvent can be possible in the absenceof His-141, which could account for the significant residual catalyticactivity of His-141→Asn arginase. A proton shuttle function for His-141of arginase is analogous to that recently reviewed for His-64 of thezinc metalloenzyme carbonic anhydrase II (Christianson et al., 1996,Acc. Chem. Res. 29:331-339).

As depicted in FIG. 3, charged amino acid residues are located onopposite sides of the active-site cleft, near the surface of thearginase trimer. The presence of these charged residues can contributeto the exquisite specificity of substrate recognition by arginase.Structure-activity relationships using arginine analogs indicate thatelectrostatic interactions of arginase with the alpha-substituents ofthe substrate are critical for catalysis. Deletion of thealpha-carboxylate group or the alpha-amino group of arginine results in10²-10⁵-fold reductions in k_(cat)/K_(m) (Reczkowski et al., 1994, Arch.Biochem. Biophys. 312:31-37). Inspection of the arginase active siteindicates that the positively charged side chain of Arg-21 interactswith the negatively charged alpha-carboxylate group of the substrate,and that the negatively charged side chain of Asp-181 interacts with thepositively charged alpha-amino group of the substrate. A model ofarginine binding to the active site of arginase is illustrated in FIG.4.

A model of arginine hydrolysis by arginase is illustrated in FIG. 5. Theionization of metal-bound water probably reflects the apparent pK_(a)value of 7.9 observed in the pH rate profile of the enzyme (Kuhn et al.,1991, Arch. Biochem. Biophys. 286:217-221). In the first step of thehydrolytic mechanism, Asp-128 stabilizes the metal-bridging hydroxideion with a hydrogen bond, while the hydroxide ion performs anucleophilic attack at the guanidinium carbon of arginine. The resultingtetrahedral intermediate collapses upon proton transfer to the aminogroup of ornithine. This proton transfer is probably mediated byAsp-128. Subsequently, His-141 shuttles a proton from bulk solvent tothe ε-amino group of ornithine prior to dissociation of ornithine fromarginase. Upon ornithine dissociation, a water molecule displaces urea,resulting in dissociation of urea from arginase. Metal coordinationfacilitates the ionization of this water molecule to regenerate anucleophilic hydroxide ion. Proton transfer from the water molecule tobulk solvent can be mediated by His-141.

It is instructive to consider the chemical function of arginase not onlywithin the context of mammalian nitrogen metabolism, but also within thegreater context of the biosphere. In the nitrogen cycle, two bi-nuclearmetalloenzymes of different tertiary structure are prominent, namely Mn₂²⁺-arginase and Ni₂ ²⁺-urease.

The former enzyme provides for abundant release of urea into theenvironment by mammals, and the latter enzyme allows the use of thisurea as a nitrogen source by bacteria, fungi, and plants.

The recent determination of the crystal structure of Ni₂ ²⁺-ureaseobtained from the bacterium Klebsiella aerogenes (Jabri et al., 1995,Science 268:998-1004) reveals some interesting parallels between thesetwo enzymes which have evolved to catalyze urea chemistry. Both arginaseand urease comprise a bi-nuclear transition metal cluster, and bothenzymes contain roughly similar constellations of catalyticallyimportant carboxylate and imidazole groups within their respectiveactive sites. Interestingly, Mn₂ ²⁺-substituted urease exhibitscatalytic activity, albeit only 2% of that of the native Ni₂ ²⁺-urease(Park et al., 1996, Biochemistry 35:5345-5352). Preliminary results withNi₂ ²⁺-substituted arginase reveal no measurable catalytic activity.Optimal catalytic activity in each system seems to have evolved withhigh selectivity for one particular bi-nuclear metal cluster of specificcomposition and structure.

EXAMPLE 2 2(S)-Amino-6-Boronohexanoic Acid (ABHA) is an EffectiveInhibitor of Arginase

Using structural data described in Example 1, the structure of aninhibitor of arginase activity was designed which resembles the proposedtransition state intermediate of the arginine hydrolysis reactioncatalyzed by arginase. The inhibitor was made and tested and it wasdetermined to be a potent inhibitor of arginase activity.

The tetrahedral borate anion is a modest inhibitor of Mn₂ ²⁺-arginase, acritical metalloenzyme of mammalian nitrogen metabolism. The crystalstructure of the arginase-ornithine-borate complex, as described herein,reveals a net displacement of the solvent molecule bridging thebi-nuclear manganese cluster by a borate oxygen atom in the nativeenzyme active site. This binding mode is reminiscent of the tetrahedralintermediate proposed for arginase-catalyzed arginine hydrolysis, asdescribed in Example 1 herein. ABHA, a boronic acid-based arginineisostere appears to bind to arginase as the tetrahedral boronate anionand mimic the conformation of the tetrahedral arginine-hydrolysisintermediate.

ABHA was synthesized and the ability of ABHA to inhibitarginase-catalyzed arginine hydrolysis was evaluated. ABHA is one of themost potent reversible inhibitors of arginase described to date, havingan IC₅₀ value of 0.8 micromolar. Complete kinetic characterization ofABHA was complicated by non-linearity of unknown origin, there being noevidence for slow-binding behavior. Competition binding experimentsusing N-hydroxy-arginine indicate that K_(d) for ABHA was less than orequal to 0.1 micromolar.

Based on analysis of the crystal structure of thearginase-ornithine-borate complex, a binding model for ABHA waspostulated, in which the metal-bridging solvent molecule observed in thenative enzyme is displaced by an oxygen atom of the tetrahedral boronicacid anion. Presumably, the boronic acid moiety of ABHA effectsinhibition of the enzyme by displacing the metal-bridging solventmolecule in a similar manner.

The materials and methods used in the experiments presented in thisExample are now described.

Thin layer chromatography (TLC) was performed using Merck (Merck & Co.,West Point, Pa.) silica gel 60 F₂₅₄ glass plates. Compounds withtert-butyloxycarbonylamino groups or free amino groups were visualizedon TLC plates by applying a ninhydrin solution (comprising 0.1% (w/v)ninhydrin in 95% (v/v) n-butanol, 4.5% (v/v) water, 0.5% (v/v) glacialacetic acid) to the plates and then heating the plates until colorevolved.

Column chromatography was performed using a column packed with Merck(Merck & Co., West Point, Pa.) silica gel 60 (230-240 mesh ASTM) underpositive nitrogen pressure.

Melting points were determined visually in an open capillary on a ThomasHoover capillary melting point apparatus.

¹H-NMR and ¹¹B-NMR spectra were measured using a Bruker AC-250 (250megahertz) NMR spectrometer and a Bruker AC-200 (200 megahertz) NMRspectrometer, respectively. Chemical shifts were expressed in parts permillion (ppm) and referenced either CDCl₃ or CD₃OD.

Arginase activity was assessed using the ⁽¹⁴C-guanidino)-arginine assayof Rüegg et al. (1980, Anal. Biochem. 102:206-212), as described(Reczkowski et al., 1994, Arch. Biochem. Biophys. 312:31-37). In initialexperiments, IC₅₀ values for ABHA and N-hydroxyarginine (K_(i)=42micromolar; Daghigh et al., 1994, Biochem. Biophys. Res. Commun.202:174-180) were compared. Assays were performed in 100 millimolarCHES, pH 9.0, containing 1 millimolar arginine.

2(S)-N-(tert-butyloxycarbonyl)-glutamic acid tert-butyl ester (compound1 in FIG. 6) was obtained from Sigma Chemical Company (St. Louis, Mo.).Triethylamine and DMSO (dimethyl sulfoxide) were obtained from FisherScientific (Malvern, Pa.). All other reagents were obtained from AldrichChemical Co. (St. Louis, Mo.).

The chemical synthetic scheme used to make ABHA is illustrated in FIG.6.

Synthesis of 2(S)-N-(tert-butyloxycarbonyl)-5-hydroxypentanoic acid,tert-butyl ester (Compound 2 in FIG. 6)

To a solution of 2.339 grams (7.71 millimoles)2(S)-N-(tert-butyloxycarbonyl)-glutamic acid tert-butyl ester in 70milliliters of tetrahydrofuran (THF) maintained at −5° C. was added 7.80milliliters (77.1 millimoles) triethylamine and 7.37 milliliters (77.1millimoles) ethyl chloroformate. The mixture was maintained at −5° C.for ten minutes with stirring. The precipitant triethylamine salt wasquickly removed by filtration. The filtrate was added to a suspension ofsodium borohydride in 10 milliliters of water and stirred at roomtemperature overnight. The resulting crude product was purified bycolumn chromatography on silica gel, using 2:1 hexane:ethyl acetate asan eluent, to yield 2(S)-N-(tert-butyloxycarbonyl)-5-hydroxypentanoicacid, tert-butyl ester (“Compound 2”).

Synthesis of Compound 3 in FIG. 6

Compound 3 was made by Swem oxidation of Compound 2, as described(Nancuso et al., 1978, J. Org. Chem. 43:2480-2482), and was useddirectly without purification.

Synthesis of Compound 4 in FIG. 6

Compound 4 was made by subjecting crude Compound 3 to a Wittig reactionas described (Bowden, 1975, Synthesis 784) using triphenylphosphoniummethylide. Compound 3, 1.151 grams (4.01 millimoles), was dissolved inTHF and was added directly to the triphenylphosphonium methylidesolution. The Wittig reaction was stirred and allowed to proceed forfifteen minutes at −78° C. and then overnight at room temperature.Compound 4 was purified by separating the Wittig reaction products usinga silica gel column using 10:1 hexane:ethyl acetate as an eluent, andthen repeating the separation to improve purity. Following concentrationin vacuo, 450 milligrams (22% over two steps) of product as a colorlessoil was obtained. TLC using 3:1 hexane:ethyl acetate as the mobile phaseindicated a single spot at R_(f)=0.60. Compound 4 had the followingproperties: ¹H-NMR (CDCl₃) δ 5.80 (m, 1H), 5.00 (m, 3H), 4.15 (m, 1H),2.05 (m, 2H), 1.85 (m, 1H), 1.65 (m, 1H), 1.45 (s, 9H), 1.40 (s, 9H).

Synthesis of2(S)-N-(tert-butyloxycarbonyl)-6-[(1S,2S,3R,5S)-(+)-pinanedioxaboranyl]-hexanoicacid, tert-butyl ester (Compound 5 in FIG. 6)

To a stirred solution of 6.05 milliliters (6.05 millimoles) of 1 molarBH₃. THF in 60 milliliters THF at 0° C. was added 345 milligrams (1.21millimoles) of Compound 4. The reaction mixture was stirred for twohours at room temperature and monitored for the disappearance ofCompound 4 by performing TLC, using 3:1 hexane:ethyl acetate as themobile phase. Excess non-reacted borane was quenched by slow addition ofmethanol until gas evolution, as indicated by bubbling, ceased. Thesolvent was evaporated in vacuo. The crude material was taken up inCH₂Cl₂. Excess (1S,2S,3R,5S)-(+)-pinanediol (1 gram, 5.87 millimoles)was added to the solution of crude material in CH₂Cl₂. Esterificationwith (+)-pinanediol was allowed to proceed at room temperature for twohours. Compound 5 was purified by column chromatography using silicagel, using 25:1 hexane:ethyl acetate as an eluent. Compound 5 wasobtained as an oil in 31% yield (174 milligrams) following concentrationin vacuo. TLC, using 10:1 hexane:ethyl acetate as the mobile phase,indicated a single spot at R_(f)=0.15. Compound 5 had the followingproperties ¹H-NMR (CDCl₃) δ 4.95 (d, 1H), 4.20 (d, 1H), 4.1 (m, 1H),2.40-2.15 (m, 2H), 2.05 (m, 1H), 1.95-1.65 (m, 411), 1.60 (s, 3H), 1.42(2s, 18H), 1.35 (apparent s, 4H), 1.25 (s, 3H), 1.05 (d, 1H), 0.80 (m,5H). The molecular mass calculated for C₂₅H₄₄NO₆B+H is 466.33. Themolecular mass of Compound 5 observed using ZAB-E CI+/MS massspectrometry was 466.25.

Synthesis of 2(S)-amino-6-boronohexanoic Acid, Hydrochloride Salt (ABHA;Compound 6 in FIG. 6)

To a stirred solution of 174 milligrams of Compound 5 in CH₂Cl₂ at −78°C. was slowly added 1.49 milliliters of 1 molar BCl₃ (1.49 millimoles)in CH₂Cl₂. The reaction mixture was stirred for fifteen minutes at −78°C. and then for thirty minutes at 0° C. The solvent CH₂Cl₂ and excessBCl₃ were evaporated by passing a flow of nitrogen gas over the mixture.Continued drying with N₂ gas afforded crude product as an orange solid.Addition of acetonitrile removed most of the color from the crudeproduct. Vacuum filtration followed by several washings withacetonitrile yielded a pale pink-colored solid which, upon air-drying,had a faint discoloration. Acetone was added to the pale pink-coloredsolid. Vacuum filtration followed by several washings with acetoneremoved all discoloration from the solid to yield 33 milligrams (42%yield) of pure ABHA as a white solid. TLC, using 80:10:10n-butanol:acetic acid:water as the mobile phase, indicated a single spotat R_(f)=0.15. ABHA had a melting point between 148 and 150° C.,dec.>130°, and the following properties: ¹H-NMR (CD₃OD) δ 3.95 (t, 1H),1.90 (m, 2H), 1.45 (m, 4H), 0.82 (m, 2H); ¹¹B NMR (CD₃OD) δ 18.86 (s).

Crystallization and X-ray Crystal Structure Determination of ABHA

In a 3.7 milliliter screw-cap glass vial, 11 milligrams of ABHA weredissolved in 0.200 milliliters of ethanol. The screw-cap on the glassvial was not tightened, in order to allow slow evaporation of ethanol.The vial was placed in a fume hood for two days. Upon inspection, allsolvent had evaporated and ABHA had crystallized as needles and plates.A section of one of the plates proved suitable for crystallographicstudies. Larger crystals were grown by slow evaporation of 11 milligramsABHA dissolved in 0.200 milliliters of water over a period of five days.

ABHA, which has the molecular formula C₆H₁₅BNO₄Cl, crystallizes in theorthorhombic space group P2₁2₁2₁ (systematic absences h00: 1=odd; 0k0:k=odd; 001: 1=odd), with a=9.914(2) angstroms, b=20.213(2) angstroms,c=5.1801(6) angstroms, V=1038.0(2) angstroms³, Z=4 and d_(calc)=1.353grams per cubic centimeter. X-ray intensity data were collected using aRigaku R-AXIS IIc area detector employing graphite-monochromatedMo-K_(α) radiation (λ=0.71069 angstrom) at a temperature of 295°K.Indexing was performed from a series of 1° oscillation images, withexposures of thirty minutes per frame. A hemisphere of data wascollected using 8° oscillation angles, with exposures of sixty minutesper frame and a crystal-to-detector distance of 82 millimeters.Oscillation images were processed, using the bioteX program, to producea listing of non-averaged F² and σ(F²) values which were provided to aSilicon Graphics Indigo R4000 computer for further processing andstructure solution, using the teXsan program. A total of 4539reflections were measured over the ranges: 5.76 ≦2θ≦50.70°, −10≦h≦11,−21≦k≦24, −6≦1≦6, yielding 1838 unique reflections (R_(int)=0.0590). Theintensity data were corrected for Lorentz and polarization effects, butnot for absorption.

The structure was solved by direct methods, using the SIR92 program.Refinement was by fall matrix least squares, based on F² using theSHELXL-93 program. All reflections were used during refinement. Valuesof F² that were experimentally negative were replaced by F²=0. Theweighting scheme used was w=1/[σ²(F_(o) ²)+0.0537P²+2.4010P], whereP=(F_(o) ²+2F_(c) ²) 3. Non-hydrogen atoms were refined anisotropically,and hydrogen atoms were refined according to a “riding” model in whichthe positions of the hydrogen atoms are re-idealized before each leastsquares cycle by applying the coordinate shifts of the atom to whicheach hydrogen is attached. Refinement converged to R₁=0.0768 andwR₂=0.1627 for 1580 reflections for which F>4σ(F) and R₁=0.0905,wR₂=0.1756 and GOF=1.106 for all 1838 unique, non-zero reflections and122 variables. The maximum Δ/σ in the final cycle of least squares was0.002 and the two most prominent peaks in the final difference Fourierwere +0.351 and −0.514 e/Å³.

Crystal Stricture of the Arginine-Ornithine-Borate Complex

Crystals of rat liver arginase were prepared as described (Kanyo et al.,1992, J. Mol., Biol. 224:1175-1177) and transferred to a buffer solutioncomprising 10 millimolar ornithine and 10 millimolar sodium borate.X-ray diffraction data at 3.0 angstroms resolution were collected andprocessed as described above. 28,047 total reflections, and 13,114unique reflections (9-3 angstroms) were used in refinement, which was74% complete, with R_(merge)=0.062.

The atomic coordinates of native rat liver arginase, as described hereinin Example 1, served as the starting model for refinement of thestructure of the arginase-ornithine-borate complex using the X-PLORprogram (Brunger et al., 1987, Science 235:458-460). Refinement of thestructure of the complex converged smoothly to a final crystallographicR factor of 0.190 for 9-3 angstrom data (R_(free)=0.301), withroot-mean-square deviations from ideal bond lengths and angles of 0.013angstrom and 1.6°, respectively.

Other methods which were used but not described herein are well knownand within the competence of one of ordinary skill in the art ofchemical synthesis and molecular biology.

The results obtained in the experiments presented in this Example arenow described.

Table 2 lists cell information, data collection parameters, andrefinement data used to determine the structure of ABHA. Finalpositional and equivalent isotropic thermal parameters are given inTable 3. Anisotropic thermal parameters are in Table 4. Tables 5 and 6list bond distances and bond angles. A representation of the molecularstructure of ABHA, made using the ORTEP program, is depicted in FIG. 7.

TABLE 2 Summary of Structure Determination of ABHA Formula: C₆H₁₅BNO₄ClFormula weight: 211.45 Crystal class: Orthorhombic Space group: P2₁2₁2₁(#19) z 4 Cell constants: a, Å 9.914(2) b, Å 20.213(2) c, Å 5.1801(6) V,Å³ 1038.0(2) μ, centimeters⁻¹ 3.52 crystal size, millimeters 0.15 × 0.05× 0.005 D_(calc), grams per cubic centimeter 1.353 F(000) 448 Radiation:Mo-K_(α) (λ = 0.71069 Å) 2θ range 5.76-50.70° hkl collected: −10 ≦ h ≦11; −21 ≦ k ≦ 24; −6 ≦ 1 ≦ 6 No. reflections measured: 4539 No. uniquereflections: 1838 (R_(int) = 0.0590) No. observed reflections 1580 (F >4σ) No. reflections used in refinement 1838 No. parameters 122 R indices(F > 4σ) R₁ = 0.0768 wR₂ = 0.1627 R indices (all data) R₁ = 0.0905 wR₂ =0.1756 GOF 1.106 Final Difference Peaks, e/Å³ +0.351, −0.514

TABLE 3 Refined Positional Parameters for ABHA Atom x y z Ueq, Å² C10.5824(2) 0.58210(8) 0.7367(3) 0.0481(4) O1 0.5795(5) 0.8844(2)0.4197(8) 0.0503(11) H1 0.614(3) 0.898(3) 0.286(8) 0.075 O2 0.4088(5)0.9503(2) 0.2973(7) 0.0462(11) O3 0.3464(5) 0.5523(2) 1.3516(8)0.0451(11) H3 0.394(6) 0.5711(6) 1.457(8) 0.068 O4 0.2110(5) 0.5741(2)0.9914(8) 0.0428(10) H4 0.206(6) 0.5337(4) 1.002(9) 0.064 N1 0.2768(5)0.9342(2) 0.7515(9) 0.0377(11) H1a 0.238(3) 0.923(2) 0.900(5) 0.056 H1b0.218(2) 0.929(2) 0.623(5) 0.056 H1c 0.3019(8) 0.9765(3) 0.759(9) 0.056C1 0.4592(6) 0.9135(3) 0.4522(10) 0.0351(13) C2 0.3974(6) 0.8919(2)0.7051(10) 0.0336(13) H2 0.4628(6) 0.8988(2) 0.8444(10) 0.045 C30.3579(7) 0.8183(2) 0.6964(10) 0.043(7) H3a 0.2711(7) 0.8142(2)0.6113(10) 0.057 H3b 0.4237(7) 0.7946(2) 0.5930(10) 0.057 C4 0.3491(8)0.7856(3) 0.9608(11) 0.043(2) H4a 0.2794(8) 0.8072(3) 1.0617(11) 0.058H4b 0.4342(8) 0.7912(3) 1.0506(11) 0.058

TABLE 4 Refined Thermal Parameters (U's) for ABHA Atom U₁₁ U₂₂ U₃₃ U₂₃U₁₃ U₁₂ C1 0.0505(9) 0.0562(8) 0.0377(7) −0.0026(8) −0.0056(8) 0.0058(7)O1 0.044(3) 0.062(3) 0.044(3) 0.013(2) 0.013(2) 0.015(2) O2 0.053(3)0.048(2) 0.037(2) 0.010(2) 0.007(2) 0.002(2) O3 0.053(3) 0.035(2)0.047(3) 0.008(2) −0.013(2) −0.003(2) O4 0.055(3) 0.033(2) 0.041(2)−0.001(2) −0.012(2) −0.003(2) N1 0.044(3) 0.037(2) 0.032(2) 0.003(2)0.006(2) −0.003(2) C1 0.046(3) 0.033(3) 0.027(3) 0.000(3) 0.003(2)−0.005(3) C2 0.044(3) 0.032(2) 0.025(3) 0.000(2) 0.004(3) 0.004(2) C30.070(4) 0.028(3) 0.030(3) 0.004(2) 0.004(3) −0.003(3) C4 0.072(4)0.027(3) 0.032(3) 0.004(2) 0.001(3) −0.002(3) C5 0.074(5) 0.034(3)0.038(3) 0.006(3) −0.007(3) −0.004(3) C6 0.057(4) 0.033(3) 0.040(4)0.003(3) −0.004(3) −0.007(3) B1 0.047(4) 0.029(3) 0.033(3) 0.002(3)−0.003(3) −0.001(3) The form of the anisotropic displacement parameteris: exp[−2π²(a*²U₁₁h² + b*²U₂₂k² + c*²U₃₃l² + 2b*c*U₂₃kl + 2a*c*U₁₃hl +2a*b*U₁₂hk)].

TABLE 5 Bond Distances in ABHA Bond Bond Distance, Å O1-C1 1.342(7)O4-B1 1.383(8) C2-C3 1.539(7) C5-C6 1.522(8) O2-C1 1.203(6) N1-C21.491(7) C3-C4 1.523(7) C6-B1 1.568(8) O3-B1 1.363(7) C1-C2 1.511(7)C4-C5 1.530(7)

TABLE 6 Bond Angles in ABHA Bond Angle, Bond Angle, Pair degrees Pairdegrees O2-C1-O1 123.9(5) C4-C3-C2 114.1(5) O2-C1-C2 126.1(5) C3-C4-C5111.7(5) O1-C1-C2 110.0(5) C6-C5-C4 114.8(5) N1-C2-C1 107.4(4) C5-C6-B1116.5(5) N1-C2-C3 110.8(5) O3-B1-O4 116.9(5) C1-C2-C3 111.0(4) O3-B1-C6123.7(5) O4-B1-C6 119.3(5)

Arginine hydrolysis involves a metal-activated solvent molecule thatsymmetrically bridges the Mn²⁺—Mn²⁺ ion pair in the native enzyme. Thereaction coordinate of hydrolysis is postulated to proceed through atetrahedral intermediate resulting from nucleophilic attack of themetal-bridging hydroxide ion at the guanidinium carbon of arginine, asdepicted in FIG. 8A (Kanyo et al., 1996, Nature 383:554-557).

Ornithine and borate are known to be relatively weak inhibitors ofarginase activity. The simultaneous presence of both ornithine andborate inhibits arginase activity more that the presence of eithercompound alone. In order to understand the mode of inhibition, the X-raycrystal structure of the ternary arginase-ornithine-borate complex wasdetermined. The tetrahedral borate anion mimics binding interactionspostulated for the tetrahedral transition states in the physiologicalarginine-hydrolysis reaction, as depicted in FIG. 8A (Kanyo et al.,1996, Nature 383:554-557). The high affinity of ABHA for arginaseapparently results from the structural similarity between the hydratedform of ABHA, as depicted in FIG. 8B, and the proposed tetrahedralintermediate and flanking transition states for arginase-catalyzedarginine hydrolysis, as depicted in FIG. 8A.

Crystal Structure of the Arginase-Ornithine-Borate Complex

The crystal structure of the arginase-ornithine-borate complex revealednet displacement of the manganese-bridging solvent molecule of thenative enzyme by an oxygen of the tetrahedral borate anion, as depictedin FIG. 9. No other structural changes are observed in the manganesecoordination polyhedra, and the average metal-metal separation distanceis 3.5 angstroms, as predicted from EPR studies (Khangulov et al., 1995,Biochemistry 34;2015-2025). The average metal-metal separation is 3.3angstroms in the native enzyme (Kanyo et al., 1996, Nature 383:554-557).

Although the low resolution of this structure determination precludes adefinitive conclusion on the binding conformation of ornithine, aninteraction between the alpha-carboxylate group of ornithine and theside chain of Arg-21 is evident in two of the three arginase subunits.The interaction between Arg-21 and the carboxylate group is consistentwith the structure-based proposal for arginase-substrate recognition, asdescribed herein in Example 1. Using the crystal structure of thecomplex determined by this experiment, a structure for a boronicacid-based arginase analog, ABHA, was designed.

Evaluation of the Inhibitory Potential of 2(S)-Amino-6-BoronohexanoicAcid (ABHA)

Boronic acids are effective aminopeptidase and serine proteaseinhibitors because they presumably bind as tetrahedral transition stateanalogs (Baker et al., 1980, Fed. Proc., Fed. Am. Soc. Exp. Biol.39:1686; Matteson et al., 1981, J. Am. Chem. Soc. 103:5241-5242; Bakeret al., 1983, Biochemistry 22:2098-2103; Kettner et al., 1984, J. Biol.Chem. 259:15106-15114; Shenvi, 1986, Biochemistry 25:1286-1291). Theelectron-deficient boron atom of a boronic acid promotes addition to theboron atom of a suitable nucleophile, such as a protein-boundnucleophile or a solvent molecule, yielding a stable anionnictetrahedral specices. Based on the structure of the ternaryarginase-ornithine-borate complex described herein, it was postulatedthat the boronic acid analog of arginine, 2(S)-amino-6-boronohexanoicacid (ABHA), would bind avidly to arginase as the hydrated anion tomimic the tetrahedral intermediate and its flanking transition states(see FIG. 8). ABHA is the first example of a boronic acid-based arginineisostere.

Determination of the crystal structure of ABHA confirmed the presence ofthe expected trigonal planar geometry of the boronic acid moiety ofABHA.

The tetrahedral borate anion is a modest, noncompetitive inhibitor ofarginase, having a K_(i) value of 1.0 millimolar and a _(Ki) value of0.26 millimolar. Inhibition of arginase activity by borate is even morepronounced in the presence of ornithine, which is a competitiveinhibitor of arginase, having a K_(i) value of 1.0 millimolar (Pace etal., 1981 Biochem. Biophys. Acta 658:410-412; Reczkowski et al., 1994,Arch. Biochem. Biophys. 312:31-37; Khangulov et al., 1995, Biochemistry34:2015-2025). Measurement of the inhibitory potential of arginaseinhibitors yielded an IC₅₀ value of 80 micromolar for N-hydroxyarginineand an IC₅₀ value of 0.8 micromolar for ABHA.

A more complete kinetic analysis of inhibition of arginase by ABHA wascomplicated by non-linearity of kinetic re-plots. The origin of thisnon-linearity is not clear, since ABHA is a reversible inhibitor thatexhibits no evidence of slow-binding behavior. Additional evidence forhigh affinity binding of arginase and ABHA was derived from competitionbinding experiments using ABHA and N-hydroxyarginine, as monitored byfluorescence spectroscopy. Addition of N-hydroxyarginine to a solutionof arginase results in a significant decrease in intrinsic proteinfluorescence at 327 nanometers. Addition of a saturating concentrationof ABHA to the arginase-N-hydroxyarginine complex restores thefluorescence of the enzyme to that observed for the enzyme alone.Addition of ABHA alone to arginase does not result in significantchanges in protein fluorescence. These experiments indicate thatK_(d)≦0.1 micromolar for ABHA at pH 7.5 and at pH 9.0.

ABHA is one of the most potent inhibitors of Mn₂ ²⁺-arginase reported todate. Previously reported inhibitors include various free amino acids(millimolar K_(i) values), N-hydroxyarginine (K_(i)=42 micromolar),N-hydroxyindospicine (K_(i)=20 micromolar), N-hydroxylysine (K_(i)=4micromolar), and N-hydroxy-nor-arginine (K_(i)=0.5 micromolar, Daghighet al., 1994, Biochem. Biophys. Res. Commun. 202:174-180; Hunter et al.,1946, J. Biol. Chem. 157:427-446; Boucher et al., 1994, Biochem.Biophys. Res. Commun. 203:1614-1621; Custot et al., 1996, J. Biol.Inorg. Chem. 1:73-82; Custot et al., 1997, J. Am. Chem. Soc.119:4086-4087). It is known that the closer the structural analogybetween an inhibitor and the catalytic transition state, the tighter theinhibitor is expected to bind (Pauling, 1946, Chem. Eng. News24:1375-1377; Wolfenden, 1969, Nature 223:704-705; Wolfenden, 1976,Annu. Rev. Biophys. 5:271-306). It appears that the high affinity ofarginase for ABHA arises from the fact that the hydrated form of ABHA isthe closest structural analog of the tetrahedral intermediate of thearginase-catalyzed arginine hydrolysis reaction, and its flankingtransition states, generated to date.

EXAMPLE 3

Additional Boronic Acid-Based Arginine Analogs Inhibit Arginase withHigh Affinities and Unusual Binding Kinetics

Boronic acid-based and trihydroxysilyl-based transition state analoginhibitors for arginase have been designed, synthesized, and evaluated.Initial characterization of the inhibitory potency of these compoundswas achieved using a new chromogenic arginase substrate,1-nitro-3-guanidinobenzene (compound 18). Surprisingly, only thetrihydroxysilyl-based inhibitor, S-(2-trihydroxysilylethyl)-cysteine(compound 16), yielded linear kinetics with a modest K_(i) of 420micromolar (assuming competitive inhibition). Due to non-linearity ofkinetic re-plots, the two most potent boronic acid-based inhibitors,2(S)-amino-6-boronohexanoic acid (ABHA; compound 7) andS-(2-boronoethyl)-L-cysteine (compound 15), were further characterizedby assay with [¹⁴C-guanidino]-L-arginine and by the technique oftitration calorimetry. The K_(d) values obtained by titrationcalorimetry for compounds 7 and 15 were 0.11 micromolar and 2.22micromolar, respectively.

The enzymes of arginine catabolism have been the subject of increasinglyintense research interest. Of particular interest are the criticalmacrophage enzymes, arginase and nitric oxide (NO) synthase. Arginasecatalyzes hydrolysis of L-arginine to form L-ornithine and urea(Christianson, 1997, Prog. Biophys. Molec. Biol. 67:217-252), and NOsynthase catalyzes oxidation of L-arginine to form citrulline and NO(Griffith et al., 1995, Annu. Rev. Physiol. 57:707-736). Intriguingly,these two enzymes are reciprocally regulated at the level oftranscription (Modolell et al., 1995, Eur. J. Immunol. 25:1101-1104;Wang, et al., 1995, Biochem. Biophys. Res. Commun. 210:1009-1016) and atthe level of catalytic activity (Corraliza et al., 1995, Biochem.Biophys. Res. Commun. 206:667-673). N^(ω)-Hydroxy-L-arginine is anintermediate in the NO synthase reaction (Pufahl et al., 1992,Biochemistry 31:6822-6828; Stuehr et al., 1991, J. Biol. Chem.266;6259-6263; Klatt et al., 1993, J. Biol. Chem. 268:14781-14787;Campos et al., 1995, J. Biol. Chem. 270:1721-1728; Pufahl et al.,Biochemistry 34:1930-1941); significant concentrations ofN^(ω)-hydroxy-L-arginine appear to dissociate from NO synthase to serveas an endogenous competitive inhibitor of arginase with K_(i)=42micromolar (Chenais et al., 1993, Biochem. Biophys. Res. Commun.196:1558-1565; Buga et al., 1996, Amer. J. Physiol. 271:H1988-H1998;Daghigh et al., 1994, Biochem. Biophys. Res. Commun. 202:174-180;Boucher et al., 1994, Biochem. Biophys. Res. Commun.203;1614-16210.

Since arginase can regulate NO synthase activity by depleting cellularconcentrations of arginine (Griffith et al., 1995, Annu. Rev. Physiol.57:707-736), the two enzymes are reciprocally coordinated at the levelof enzyme activity (FIG. 11). Selective inhibition of arginase or NOsynthase might therefore result in beneficial physiological effects dueto the modulation of macrophage function.

In the present Example, various boronic acid-based arginine analogs weretested for their ability to be selective inhibitors of arginase. Thebi-nuclear manganese cluster of arginase functions to activate abridging hydroxide ion for attack at the guanidinium group of thesubstrate arginine (Reczkowski et al., 1992, J. Am. Chem. Soc.114:10992-10994; Kanyo et al., 1996, Nature 383:554-557). A keytetrahedral intermediate results (FIG. 11), and boronic acid-basedinhibitor ABHA (compound 7) wag designed to mimic this intermediate andits flanking transition states (Example 2). Specifically, theelectron-deficient boron atom of the boronic acid moiety is sufficientlyelectrophilic to facilitate addition of a catalytic nucleophile, such asa protein atom or a solvent molecule. Similar design strategies haveresulted in potent boronic acid-based inhibitors of serine proteases(Matteson et al., 1981, J. Am. Chem. Soc. 103:5241-5242; Kettner et al.,1984, J. Biol. Chem. 259: 15106-15114) and aminopeptidases (Shenvi 1986,Biochemistry 25:1286-1291). Accordingly, ABHA (compound 7) is one of themost potent boronic acid-based inhibitors of arginase known to date withIC₅₀=0.8 micromolar, yet does not inhibit NO synthase (Example 2).

Surprisingly, ABHA (compound 7) exhibited substantial non-linearity inkinetic re-plots (Example 2), and a full description of its bindingkinetics has been reported herein. These unusual binding kinetics canarise from the unique chemistry of the electron-deficient boronic acidmoiety. In order to complement this work and further probe the unusualchemistry of boronic acid-based analogs of arginine, we also report thesynthesis and kinetic evaluation of 2(S)-amino-5-boronopentanoic acid(compound 12), and the kinetic evaluation ofS-(2-boronoethyl)-L-cysteine (compound 15) (the synthesis of compound 15is previously reported; Matteson et al., 1964, J. Med Chem. 7:640-643).Additionally, the synthesis and kinetic evaluation of atrihydroxysilane-based arginine analog,S-(2-trihydroxysilylethyl)-cysteine (compound 16), as well as a newchromogenic substrate which allows for a continuous assay of arginaseactivity is reported herein.

The Materials and Methods used in this Example are now described.

General Methods

Thin layer chromatography (TLC) was performed on Merck silica gel 60F₂₅₄ glass plates. Compounds with tert-butyloxycarbonylamino groups orfree amino groups were visualized on TLC plates by first dipping theplates in a ninhydrin solution (0.1% ninhydrin in 95% n-butanol, 4.5%water, 0.5% glacial acetic acid) and then heating the plates until colorevolved. Column chromatography was performed on Merck silica gel 60(230-240 mesh ASTM) under positive nitrogen pressure. ¹H-NMR and ¹¹B-NMRspectra were recorded on a Bruker AC-250 (250 megahertz) NMR and on aBruker AC-200 (200 megahertz) NMR, respectively (¹H chemical shifts arereferenced to CHCl₃, HDO, or DMSO, and ¹¹B chemical shifts arereferenced to BF₃ diethyl ether. Electrospray mass spectrometry wasconducted by personnel in the Mass Spectrometry Center, University ofPennsylvania.

2(S)-N-(tert-butyloxycarbonyl)-aspartic acid tert-butyl ester (1) waspurchased from Bachem. Triethylamine and DMSO (dimethyl sulfoxide) werepurchased from Fisher. All other reagents were purchased from Aldrichand used without further purification.

2(S)-amino-6-boronohexanoic acid (ABHA)

The synthesis and preliminary evaluation of this boronic-based analog isdescribed in Example 2.

2(S)-N-(tert-butyloxycarbonyl)-4-hydroxypentanoic acid, tert-butyl ester(compound 8)

To a solution of 2.90 grams (10.0 millimoles) of2(S)-N-(tert-butyloxycarbonyl)-aspartic acid, tert-butyl ester in 70milliliters of THF maintained at −5° C. was added 2.79 milliliters (20.0millimoles) triethylamine and 1.92 milliliters (20.0 millimoles) ethylchloroformate. The mixture was maintained at −5° C. for five minuteswith stirring. The precipitant triethylamine salt was quickly removed byfiltration. The filtrate was added to a suspension of sodium borohydridein 5 milliliters of water and stirred at room temperature overnight. Theresulting crude product was purified by column chromatography on silicagel (with 5:1 hexane:ethyl acetate as an eluent). Followingconcentration in vacuo, 1.44 grams of product was obtained (52% yield).TLC (1:1 hexane:ethyl acetate) indicated a single spot (R_(f)=0.41).¹H-NMR (CDCl₃): δ 1.45 (s, 18H), 2.15 (m, 2H), 3.70 (m, 2H), 4.40 (m,1H), 5.40 (d, 1H).

2(S)-N-(tert-butyloxycarbonyl)-4-oxobutanoic acid, tert-butyl ester(compound 9)

To a solution of 1.83 milliliters (20.9 millimoles) of oxalyl chloridein 30 milliliters CH₂Cl₂ maintained at −78° C. was added 2.97milliliters (41.9 millimoles) of DMSO. The solution was stirred at −78°C. for 5 minutes. A solution of 1.44 grams (5.24 millimoles) of compound8 dissolved in 10 milliliters of CH₂Cl₂ was prepared, added by cannulato the pre-formed Swem reactant, and stirred at −78° C. for 15 minutes.The Swern oxidation was completed by the addition of 0.726 milliliter(14.6 millimoles) of triethylamine. The mixture was stirred for 5 min.at −78° C. and then warmed to room temperature. The CH₂Cl₂ wasevaporated in vacuo. The crude material was taken up in THF and theprecipitant triethylamine salt was removed by filtration. The materialwas purified by column chromatography on silica gel (with 6:1hexane:ethyl acetate as an eluent). The product (980 milligrams) wasobtained in 69% yield. TLC (1:1 hexane:ethyl acetate) and staining withninhydrin indicated a single spot (R_(f)=0.65) following concentrationin vacuo. ¹H-NMR (CDCl₃): δ 1.45 (s, 18H), 3.0 (t, 2H), 4.45 (m, 1H),5.35 (d, 1H), 9.75 (s, 1H).

2(S)-N-(tert-butyloxycarbonyl)-pent-4-enoic acid, tert-butyl ester(compound 10)

The Wittig salt triphenylphosphonium methylide was prepared by theaddition of 5.11 grams (14.3 millimoles) of potassium tert-butoxide to apartially dissolved solution of 1.61 grams (14.3 millimoles)methyltriphenylphosphonium bromide in 100 milliliters of THF maintainedat 0° C. The mixture was stirred at 0° C. for 10 minutes, and then atroom temperature for 90 minutes. The mixture turned yellow and wascooled to −78° C. The aldehyde compound 9, 0.980 gram (3.59 millimoles)dissolved in THF was added directly to the triphenylphosphoniummethylide solution. The Wittig reaction was stirred and allowed toproceed for 5 minutes at −78° C. and then overnight at room temperature.The olefin compound 10 was purified by column chromatography on silicagel (with 20: 1 hexane:ethyl acetate as an eluent). Followingconcentration in vacuo, 632 milligrams of product was obtained (65%yield). TLC (3:1 hexane:ethyl acetate) indicated a single spot(R_(f)=0.56). ¹H-NMR (CDCl₃): δ 1.40 (s, 9H), 1.45 (s, 9H), 2.50 (m,2H), 4.25 (m, 1H), 5.1 (s, 1H), 5.15 (d, 2H), 5.6-5.8 (m, 1H).

2(S)-N-(tert-butyloxycarbonyl)-5-[(1S,2S,3R,5S)-(+)-pinanedioxaboranyl]-pentanoicacid, tert-butyl ester (compound 11).

To a solution of 11.6 milliliters (11.6 millimoles) of 1 molar BH₃.THFin 10 milliliters THF, maintained at 0° C. with stirring, was added 632milligrams (2.33 millimoles) of olefin compound 10. The reaction mixturewas stirred for 15 minutes at 0° C., and then for 2 hours at roomtemperature. Excess non-reacted borane was quenched by slow addition ofmethanol until gaseous evolution, as indicated by bubbling, ceased. Thesolvent was evaporated in vacuo. The crude material was taken up inCH₂Cl₂. Excess (1S,2S,3R,5S)-(+)-pinanediol (0.793 g, 4.66 millimoles)was added to the solution of crude material in CH₂Cl₂. Esterificationwith (+)-pinanediol was allowed to proceed overnight at roomtemperature. Compound 11 was purified by column chromatography on silicagel (with 20:1 hexane:ethyl acetate as an eluent). The product wasobtained in 22% yield (226 milligrams) following concentration in vacuo.TLC (3:1 hexane:ethyl acetate) indicated a single spot (R_(f)=0.58).¹H-NMR (CDCl₃): δ 0.80 (m, 5H), 1.05 (d, 2H), 1.25 (s, 3H), 1.35 (s,3H), 1.40-1.50 (m, 20H), 1.6-1.95 (m, 3H), 2.05 (t, 1H), 2.10-2.35 (m,2H), 4.15 (m, 1H), 4.25 (d, 2H).

2(S)-amino-5-boronopentanoic acid, hydrochloride salt (compound 12)

To a solution of 226 milligrams of compound 11 in CH₂Cl₂, maintained at−78° C. chilled stirring, was slowly added 2.00 milliliters of 1 molarBCl₃ in CH₂Cl₂ (2.00 millimoles). The reaction mixture was stirred for15 minutes at −78° C. and then for 30 minutes at 0° C. The solventCH₂Cl₂ and excess BCl₃ were evaporated by allowing a flow of nitrogengas to pass over the mixture.

The product material wag taken up in acetonitrile and collected byvacuum filtration. Repeated washings with acetonitrile and then withdiethyl ether gave a crude hygroscopic solid. The solid was taken up ina minimal volume of ethanol. Precipitation of product was induced byaddition of acetone. The highly hygroscopic precipitate was collectedand removed from the filter paper by addition of ethanol. The ethanolfiltrate was collected. Ethanol was evaporated in vacuo to give 16milligrams of pure product (16% yield). TLC (80:10:10 n-butanol:aceticacid:water) indicated a single spot (R_(f)=0.096). ¹H-NMR (D₂O): δ 0.85(t, 2H), 1.55 (m, 2H), 1.95 (t, 2H), 3.95 (t, 1H).

S-(2-boronoethl)-L-cysteine, hydrochloride salt (compound 15)

This compound was synthesized as previously described (Matteson et al.,1964, J. Med Chem. 7;640-643; Matteson, 1960, J. Am. Chem. Soc. 82:4228-4233). The compound was originally synthesized and tested as awater-soluble candidate for boron neutron-capture therapy (Matteson etal., 1964, J. Med Chem. 7:640-643). TLC (80:10:10 n-butanol:aceticacid:water) of the product indicated a single spot (R_(f)=0.44). ¹H-NMR(D₂O): δ 0.95 (t, 2H), 2.50 (t, 2H), 2.75-3.05 (m, 2H), 3.72 (m, 1H).¹¹B NMR (D₂O) δ 31.33 (s).

S-(2-trihydroxysilylethy)-L-cysteine (compound 16)

A solution consisting of 3.96 grams (32.6 millimoles) L-cysteine in 50milliliters water and 5 milliliters (32.6 millimoles) of vinyltrimethoxysilane in 40 milliliters methanol was refluxed at 80° C. undera nitrogen atmosphere. At time intervals of 0, 5, and 6 hours, 32milligrams aliquots of azobisisobutyronitrile were added to thesolution. The solution was refluxed for a total of 8.5 hours at 80° C.,then stirred overnight at room temperature. The solvents, water andmethanol, were removed in vacuo. The residue was taken up in a minimalvolume of water, acidified with 1 milliliter of concentrated HCl, andthen suspended in excess acetone. After a precipitate settled, theacidic solvent was decanted and discarded. This wash with acid andacetone was repeated two more times in order to remove all non-reactedstarting material. The final precipitate was a crystalline, glass-likesolid free of contamination from non-reacted L-cysteine, which waspresent in the filtrate. The product yield was 12% (1.032 g). ¹H-NMR(D₂O): δ 0.90 (brs, 1H), 2.55 (brs, 2H), 2.95 (brs, 2H), 3.90 (brs, 1H),5.85 (brs, 3H). Electrospray mass spectroscopy indicated a strongtendency to dimerize via the silanetriol moiety. CalculatedC₅H₁₄NO₅SSi⁺, 228.04; found: 406.9, 428.8, and 444.8.

1-nitro-3-(N,N′-bis(tert-butyloxycarbonyl)guanidino)benzene (compound17)

To a stirred solution of 238 milligrams (1.72 millimoles) m-nitroanilineand 500 milligrams (1.72 millimoles)1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea in CH₂Cl₂ wasadded 438 milligrams (2.58 millimoles) of silver nitrate (Bergeron etal., 1987, J. Org. Chem. 52:1700-1703, Natsugari et al., U.S. Pat. No.4,851,422 Jul. 25, 1989; Morimoto et al., U.S. Pat. No. 4,876,251 Oct.24, 1989). A white precipitate indicative of silver methylsulfide formedafter one hour. The mixture was stirred overnight at room temperature.Compound 17 was purified by column chromatography on silica gel (with10:1 hexane:ethyl acetate as an eluent). The product was obtained in 67%yield (439 milligrams) following concentration in vacuo. TLC (3:1hexane:ethyl acetate) indicated a single spot (R_(f)=0.43). ¹H-NMR(CDCl₃): δ 1.50 (s, 9H), 1.60 (s, 9H), 7.50 (t, 1H), 7.95 (d, 1H), 8.05(d, 1H), 8.5 (s, 1H), 10.6 (s, 1H), 11.6 (s, 1H).

1-nitro-3-guanidinobenzene, hydrochloride salt (compound 18)

To a stirred solution of compound 17 (238 milligrams, 0.492 millimoles)in CH₂Cl₂ was added 1.97 milliliters of 1 molar BCl₃ (1.97 millimoles)in CH₂Cl₂. After 30 minutes, excess BCl₃ and CH₂Cl₂ were removed bypassing a stream of nitrogen gas across the surface of the solution. Theresulting material was washed with CH₂Cl₂. The product was obtained as awhite powder in 62% yield. TLC (4:1:1 n-butanol:acetic acid:water)indicated a single spot that was also UV active (R_(f)=0.47). ¹H-NMR(DMSO): δ7.6-7.8 (m, 3H), 8.0-8.15 (m, 1H), 10.25 (brs.).

Enzyme Assays

Recombinant rat liver arginase was purified as described previously(Cavalli et al., 1994, Biochemistry 33:10652-10657). Concentrations ofenzyme stock solutions were determined from absorbance at 280 nanometersusing an extinction coefficient of 1.09 milliliters per milligram percentimeter (Schimke, 1970, Methods Enzymol. 17a:311-317). The activityof wild-type arginase was monitored spectrophotometrically using aPharmacia Biotech UltroSpec™ 2000. Assays were performed in a solutioncomprising 100 micromolar MnCl₂ and 50 millimolar bicine-NaOH (pH 9.0).The compound 1-guanidino-3-nitrobenzene (i.e. compound 18) proved to bea new substrate for arginase hydrolysis, producing products urea and thechromogen m-nitroaniline. A stock solution of 200 millimolar compound 18in DMSO was prepared. A period of 15 minutes was allowed to permitcompound 18 to equilibrate with bicine buffer prior to assay. Reactionvelocities were measured at 372 nanometers, where the liberated product,m-nitroaniline, has an extinction coefficient of 1.28×10³ liters permole per centimeter. For K_(M) and k_(cat) determinations, 40microliters of 0.448 milligram per milliliter arginase was added to acuvette containing bicine buffer and the appropriate concentration ofcompound 18 (0.8-2.3 millimolar) to achieve a final volume of 1milliliter. Eadie-Hofstee plots were used to analyze the data.

Compounds 7, 12, 15, and 16 (FIG. 10, Scheme 1) were assayed using 2millimolar compound 18 as the assay substrate. The concentrations ofcompounds 7 and 15 were varied in the range 0.5-9.0 micromolar, those ofcompounds 12 and 16 were varied in the range 200-1000 micromolar. Whenactivity as a function of enzyme concentration was assayed, theconcentration of arginase trimer was varied in the range 1.1-6.4micromolar. For the boronic acid inhibitors, reciprocal plots of inversevelocity as a function of inhibitor concentration were non-linear. Forthe trihydroxysilane inhibitor, reciprocal plots of inverse velocity asa function of inhibitor concentration were linear.

Dialysis experiments of arginase with the arginase-compound 7 andarginase-compound 15 complexes were performed in following way. Thirtymicroliters of arginase at a concentration of 11.2 milligrams permilliliter were pre-incubated for thirty minutes with an appropriateconcentration of compound 7 (5 microliters of 100 micromolar stocksolution). For compound 15, 300 microliters of arginase at aconcentration of 0.938 milligram per milliliter were pre-incubated forthirty minutes with a sufficient concentration of compound 15 (30microliters of 200 micromolar stock solution) to suppress all detectableenzymatic activity. Pre-incubates were then added to bicine buffercontaining 4 millimolar compound 18. The final volume of the assayreaction solution was 1 milliliter.

The inhibition of arginase by compounds 7 and 15 was also evaluatedusing a modified version of the radioactive assay by Rüegg and Russell(Rüegg et al., 1980, Anal. Biochem. 102:206-212). Assays were performedin 100 micromolar MnCl₂, 100 millimolar CHES-NaOH (pH 9.0). Thereactions were initiated by addition of 5 microliters of a circa 1 unitper milliliter enzyme solution to 45 microliters of reaction mixturecontaining CHES buffer, 1.4 millimolar L-arginine, about 5.0×10⁴ countsper minute of L-[guanidino-¹⁴C]arginine, and variable concentrations ofthe appropriate inhibitor. After a 5-15 minute reaction time, 200microliters of a stop solution containing 7 molar urea, 10 millimolarL-arginine, and 0.25 molar acetic acid (H 4.5) was added to the reaction(arginase has essentially no activity under these conditions).[¹⁴C]-Urea was separated from non-reacted L-[guanidino-¹⁴C]arginine bytreatment with 200 microliters of a 1:1 (vol/vol) slurry of Dowex 50W-X8 in water, and quantitated by adding 200 microliters of thesupernatant from the Dowex treatment to 3 milliliters of Liquiscint™(National Diagnostics) for liquid scintillation counting in a BeckmanLS5000CE counter.

Isothermal Titration Calorimetry

All calorimetry experiments were conducted on a MCS isothermalcalorimeter from MicroCal, Inc. (Northampton, Mass.). Arginase wasexhaustively dialyzed against a solution comprising 100 micromolar MnCl₂and 50 millimolar bicine-NaOH (pH 8.5). Inhibitor was dissolved at aconcentration of 1.5 millimolar in an aliquot of the same solution.Prior to the titration experiment, samples were de-gassed under vacuumfor 5 minutes. The sample cell (effective volume 1.366 milliliters) wasover-filled with 1.8 milliliters of arginase at a concentration of0.0358 millimolar and the reference cell was filled with water. Thecontents of the sample cell were titrated with 30-40 aliquots (2.5microliters each) of inhibitor (an initial 1 microliters injection wasmade, but not used in data analysis). After each injection, the heatchange was measured and converted to the corresponding enthalpy value.The reaction mixture was continuously stirred at 400 rotations perminute during titration. Control experiments were carried out bytitrating the inhibitor into the buffer solution under identicalexperimental conditions. Data analysis was performed using Origin™software provided with the instrument. The calorimetric data arepresented with the background titrations subtracted from theexperimental data. The amount of heat produced per injection wascalculated by integration of the area under each peak. The data were fitto the following equation,

q=VΔH[E]_(t)K[L]/1+K[L],

where q is the heat evolved during the course of the reaction, V is thecell volume, ΔH is the binding enthalpy per mole of ligand, [E]_(t) isthe total enzyme concentration, K is the binding constant, and [L] isinhibitor concentration (Fisher et al., 1995, Methods Enzymol. 1995259:194-221; Wiseman et al., 1989, Anal. Biochem. 179:131-137).

The Results of the experiments presented in this Example are nowdescribed.

The K_(M) and k_(cat) values for arginase-catalyzed hydrolysis of thenew chromogenic assay substrate, 1-nitro-3-guanidinobenzene (compound18), were 1.6±0.2 millimolar and 0.09±0.02 reciprocal minutes,respectively (FIG. 2), based on a monomer molecular mass of 35kilodaltons. Within experimental error, the K_(M) value for compound 18was identical to that reported for arginine itself (K_(M)=1.4±0.3millimolar; Fisher et al., 1995, Methods Enzymol. 259:194-221; Wisemanet al., Anal. Biochem. 179:131-137). The k_(cat) value of compound 18was five orders of magnitude lower than that reported for arginine(k_(cat)=15,000±1200 reciprocal minutes; Cavalli et al., 1994,Biochemistry 33:10652-10657). Despite its low k_(cat) value, substrate18 provided a continuous chromogenic assay with no apparent backgroundand high precision. Furthermore, the synthesis of compound 18 requiredonly two straightforward steps, which facilitates the rapid productionof large quantities of this compound.

For fully competitive, non-competitive, and uncompetitive inhibitors,plots of v_(o)/v as a function of (I), should be linear (Todhunter,1979, Methods Enzymol. 63:383-411; where v_(o) is enzyme velocity in theabsence of inhibitor, v is enzyme velocity in the presence of inhibitor,and (I) is the inhibitor concentration, (E_(o)) is total enzymeconcentration). For example, this was the case for the trihydroxysilaneinhibitor compound 16, which is a fully reversible inhibitor with amodest k_(i) of 420±30 micromolar (assuming competitive inhibition; FIG.13a). However, for boronic acid inhibitor compounds 7, 12, and 15, plotsof v_(o)/v as a function of (I) were non-linear and concave upward(FIGS. 13b-13 d).

Plots of v/v_(o) versus (I) at constant (E_(o)) yielded a linearrelationship for the boronic acid inhibitors. Likewise, plots of v/v_(o)as a function of 1/(E_(o)) at constant (I) (4 micromolar ABHA) werelinear. These data lead to the observation that v/v_(o) as a function of(I)/(E_(o)) was linear for all the boronic acid inhibitors (FIG. 14).Significantly, this relationship was consistent with enzyme inactivation(Silverman, 1988, In: Mechanism-based Enzyme Inactivation: Chemistry andEnzymology, vol. I, CRC Press, Inc., Boca Raton, Fla., pp 22-23).Paradoxically, the boronic acid inhibitors gave no indication ofinactivation or time-dependent inhibition, and dialysis experimentsindicated complete and virtually instantaneous reversible inhibitionwhen enzyme and inhibitor were incubated for short time periods (30minutes), in that dialysis experiments with ABHA exhibited completelyinstantaneous reversible inhibition when arginase and ABHA pre-incubatedfor 30 minutes were diluted and assayed. Upon dilution, theconcentration of ABHA went from 14.3 micromolar to 0.5 micromolar; theconcentration of arginase went from 274 micromolar to 9.6 micromolar.The observed inhibited velocity for the diluted and assayed pre-incubatewas 0.46 micromolar per minute. A control experiment assaying theactivity of arginase at 9.6 micromolar in the presence of 0.5 micromolarABHA yielded an inhibited velocity of 0.47 micromolar per minute.

For irreversible inhibitors, the slope of linear v/v_(o) versus(I)/(E_(o)) plots represented a reciprocal turnover number: moles ofenzyme inactivated per moles of inactivator. The reciprocal slopesderived from v/v_(o) versus (I)/(E_(o)) plots gave rise to a constantdesignated the “pseudo-turnover number”, tn_(pseudo), since the boronicacids were behaving reversibly and not as inactivators. The tn_(pseudo)provides a useful means for comparing the relative inhibitor potency(Table 7). When assayed with compound 18, boronic acid inhibitors 7(ABHA) and 15 were essentially equipotent (within experimental error),with compound 7 showing a marginally lower tn_(pseudo). The tn_(pseudo)for boronic acid compound 12 was two orders of magnitude higher than thetn_(pseudo) values for compounds 7 and 15. Thus, inhibitor 12 was about400-fold less potent than compounds 7 and 15.

TABLE 7 Kinetic and Thermodynamic Data for Boronic Acid Inhibitorstn_(pseudo) (moles of inhibitor/mole K_(i) ^(a) K_(d) ^(b) Inhibitor ofarginase) (micromolar) (micromolar)  7 0.7 ± 0.1 0.1 0.11 15  1.00 ±0.004 0.7 2.22 12 420 ± 30  N.D. N.D. 16 N.A. 420 N.D. ^(a)Determined byassay with substrate 18. ^(b)Determined by titration calorimetry.

Apparent K_(i) values for compounds 7 and 15 were obtained using theradioactive [¹⁴C-guanidino]-L-arginine assay of Rüegg et al. 1980, Anal.Biochem. 102:206-212; Table 7). At low concentrations of compounds 7 and15, linear plots of v_(o)/v as a function of (I) yielded K_(i) values of0.1 micromolar and 0.7 micromolar for compounds 7 and 15, respectively(assuming competitive inhibition). It appeared that compound 7 wassignificantly more potent than compound 15 against the naturalsubstrate, L-arginine. Qualitative differences in the results ofinhibitor assays using L-arginine or compound 18 as an assay substratecan arise from structural differences between these two substrates:substrate 18 lacks an amino acid moiety and therefore might be legssensitive to displacement by the amino acid moiety of compounds 7 or 15.

The titration calorimetry data for compounds 7 and 15 reveal threeimportant insights on the inhibition of arginase by boronic acids (Table7; FIGS. 15 and 16). First, ΔH_(binding) values for compounds 7 and 15to the arginase monomer were nearly identical (−12.97 kilocalories permole and −12.75 kilocalories per mole, respectively), suggesting asimilar association mechanism. Second, the stoichiometry of inhibitorbinding was 1.07 and 0.96 per monomer for compounds 7 and 15,respectively. Finally K_(d) values (K_(d)=1/K) of 0.11 micromolar and2.22 micromolar were obtained for compounds 7 and 15, respectively.Interestingly, the results of kinetic assays agree well with titrationcalorimetry data for compound 7, with K_(i)>>K_(d). However differentassays yielded different results for compound 15, i.e., K_(j) and K_(d)values disagree by nearly an order of magnitude. Unlike K_(i), K_(d) isobtained at thermodynamic equilibrium. Prior to thermodynamicequilibrium, compound 15 may perhaps undergo a conformational change forwhich K_(i), a kinetic parameter, is less sensitive.

Boronic acid compounds 7, 12, and 15 are expected to bind with arginaseas the corresponding tetrahedral boronate anions, thereby mimicking theproposed tetrahedral intermediate and its flanking transition states forarginine hydrolysis (FIG. 17). This type of transition state analogexploits the electron-deficient nature of boron, which facilitatesaddition of a nucleophilic solvent molecule to generate the boronateanion. In solution, the neutral trigonal planar boronic acid is in a pHdependent equilibrium with the tetrahedral boronate anion. At alkalinepH values (i.e., high (HO⁻)), the tetrahedral species predominates(Anderson et al., 1964, J. Phys. Chem. 68:1128-1132); this is mostlikely the case for inhibitors binding with the arginase active site,where the local concentration of metal-bridging hydroxide is high. Byway of analogy, the tetrahedral borate anion is a non-competitiveinhibitor of arginase which exhibits a K_(II)=0.26 millimolar and aK_(IS)=0.98 millimolar, and which binds with the active site bydisplacing the metal-bridging hydroxide ion (Example 2; Pace et al.,1981, Biochim. Biophys. Acta 658:410-412; Reczkowski, R. S. Ph.D.Thesis, Temple University School of Medicine, 1990). It should be notedthat (a) the binding mode of borate, (b) the high affinities of boronicacid-based arginine analogs, and (c) the relative invariance ofsubstrate K_(M) values with perturbation of the bi-nuclear manganesecluster (Cavalli et al., 1994, Biochemistry 33:10652-10657) do notsupport a recently proposed arginase catalytic mechanism which involvesdirect arginine-manganese coordination (Khangulov et al., 1998,Biochemistry 37:8539-8550); instead, these data continue to beconsistent with the mechanism proposed by Kanyo et al. (1996, Nature383:554-557).

In contrast with the boronic acid-based arginine analogs, silanetriolcompound 16 has a fixed tetrahedral configuration, and only compound 16yielded a linear plot of v_(o)/v versus (I) (indicative of reversibleinhibition). However, arginase inhibition by compound 16 is modest:assuming competitive inhibition, K_(i)=420 micromolar. Silanetriolinhibitors have recently been developed against beta-lactamase (Curleyet al., 1997, J. Am. Chem. Soc. 119:1529-1538). Here, too, silanetriolare significantly poorer inhibitors than their boronic acid analogs.Silanetriols have additional complications as enzyme inhibitors due totheir generally poorer solubility and greater tendency to dimerize(Knight et al.,1989, J. Chem. Soc., Dalton Trans.275-281; McNeil et al.,1980, J. Am. Chem. Soc. 102:1859-1865).

Compounds 7 and 15 are among the most potent arginage inhibitorsreported to date, and the use of the new chromogenic substrate 18facilitates the rapid screening of these inhibitors. Titrationcalorimetry yields precise K_(d) values of 0.11 micromolar and 2.22micromolar for compounds 7 and 15, respectively. The unrivaled potencyand selectivity of compound 7 (ABHA) for arginase make this compoundideal for probing the reciprocal functions of arginase and nitric oxidesynthase, e.g., in the regulation of nitric oxide-induced smooth musclerelaxation as described in Example 4.

EXAMPLE 4 Biochemical and Functional Profile of Arginase Inhibitors

An increase in arginase activity has been associated with thepathophysiology of a number of conditions including an impairment innon-adrenergic and non-cholinergic (NANC) nerve-mediated relaxation ofgastrointestinal smooth muscle. It was hypothesized that an arginaseinhibitor may rectify this condition. The effects of a newly designedarginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABHA) with thecurrently available N^(ω)-hydroxy-L-arginine (L-HO-Arg) on NANCnerve-mediated internal anal sphincter (IAS) smooth muscle relaxationand the arginase activity in IAS and other tissues was compared in thisExample.

Arginase caused attenuation of IAS smooth muscle relaxations by NANCnerve stimulation that was restored by arginase inhibitors.Interestingly, L-HO-Arg, but not ABHA, caused dose-dependent andcomplete reversal of N^(ω)-nitro-L-arginine-(L-NNA)-suppressed IASrelaxation that was similar to that seen with L-arginine. Both ABHA andL-HO-Arg augmented NANC nerve-mediated relaxation of the IAS. In IAS,ABHA was about 250 times more potent than L-HO-Arg in inhibiting thearginase activity. L-HO-Arg was 10 to 18 times more potent in inhibitingthe arginase activity in the liver, as compared to that in non-hepatictissues.

It was therefore concluded that arginase has a significant role inregulating of NO synthase-mediated NANC relaxation in the IAS. Theadvent of new and selective arginase inhibitors therefore has asignificant role in discrimination of arginase isozymes and haveimportant pathophysiological and therapeutic implications ingastrointestinal motility disorders.

The purpose of the present investigation was to test a newly designedand selective arginase inhibitor, 2(S)-amino-6-boronohexanoic acid(ABHA; Example 2, compound 7) for its effectiveness in thephysiologically relevant system. The effectiveness of ABHA on arginaseactivity in IAS, rectum, brain, and liver tissues was also examined.

The Materials and Methods used in the experiments presented in thisExample are now described.

Functional Studies

Preparation of Smooth Muscle Strips

Studies were performed using circular smooth muscle strips of theinternal anal sphincter (IAS) obtained from adult opossums (Didelphisvirginiana) of either sex following pentobarbital anesthesia (40milligrams per kilogram intraperitoneally) and subsequentexsanguination. The entire anal canal was isolated carefully bydissection and transferred to a dissecting tray containing oxygenated(95% O₂ plus 5% CO₂) Krebs' solution. The composition of the Krebs'solution was as follows (in millimolar): NaCl, 118.07; KCl 4.69; CaCl₂,2.52; MgSO₄, 1.16; NaH₂PO₄, 1.01; NaHCO₃, 25 and glucose (Pufahl et al.,1995, Biochemistry 34:1930-1941; Chenais et al., 1993, Biochem. Biophys.Res. Commun. 196:1558-1565). The anal canal was cleaned of extraneousconnective tissue and blood vessels. Following this, the anal canal wasopened by making an incision along the longitudinal axis, and was pinnedflat with the mucosal side facing up. The mucosa and submucosa wereremoved by sharp dissection. Circular smooth muscle strips were obtainedfrom the whole circumference of the anal canal and divided into twoequal strips (about 1×8 millimeters). Both ends of the muscle stripswere secured with silk sutures (4-0: Ethicon Inc., Sommerville, N.J.)and used for the measurement of isometric tension.

Measurement of Isometric Tension

IAS smooth muscle strips prepared as described above were mounted ontothermogtatically-controlled 2 milliliter muscle baths (37° C.)containing oxygenated (95% O₂ and 5% CO₂) Krebs' solution. One end ofeach muscle strip was fixed to the bottom of the muscle bath with atissue holder and the other end was attached to an isometric forcetransducer (model FT03; Grass Instruments Co., Quincy, Mass.) in orderto measure isometric tension. Smooth muscle tension was recorded using aDynograph™ recorder (model R411; Beckman Instruments, Schiller Park,Ill.). After an equilibration period of 1 hour with intermittentwashings, the optimal length (L₀) and the baseline of the restingtension of each smooth muscle strip were determined as described(Mourami and Rattan, 1988, Am. J. Physiol. 255:G571-G578). Only thosesmooth muscle strips that developed spontaneous and steady tension andrelaxed in response to electrical field stimulation (EFS) were used.

Non-adrenergic Non-cholinergic (ANC) Nerve Stimulation with ElectricalField Stimulation (EFS)

EFS was delivered, via a pair of platinum wires, from a Grass stimulator(Model S88; Grass Instruments Co., Quincy, Mass.) connected in series toa Med-Lab Stimu-Splitter™ II (Med-Lab Instruments, Loveland, Colo.). TheStimusplitter™ was used to amplify and measure stimulus intensity usingoptimal stimulus parameters for the neural stimulation (12 volt, 0.5millisecond pulse duration, 200-400 milliamperes, 4 second train) atvarying frequencies of 0.5 to 20 Hertz. These parameters of EFS areknown to cause relaxation of IAS smooth muscle by selective activationof NANC myenteric neurons. Neurally-mediated relaxation of IAS smoothmuscle strips was quantified in response to different frequencies ofEFS. All the experiments were performed in the presence of atropine(1×10⁻⁶ molar) and guanethidine (3×10⁻⁶ molar).

Drug Responses

To determine the influence of arginase on NANC nerve-mediated relaxationof IAS, the effects of different doses of arginase on relaxation werefirst determined. A dose of 30 units of arginase per milliliter wasfound to be the most effective in attenuating relaxation. Theeffectiveness of arginase inhibitors (L-OH-Arg and ABHA) onarginase-induced attenuation of EFS-induced IAS relaxation was thentested. Optimal doses of arginase and L-OH-Arg in IAS have been reported(Chakder and Rattan, 1997, J. Pharmacol. Exp. Ther. 282:378-384). Inorder to determine the selectivity of arginase inhibitors in IAS, theireffects on IAS relaxation suppressed by the NO synthase inhibitor L-NNA(3×10⁵ molar) were tested. These results were compared with reversal ofrelaxation suppression caused by addition of the NO synthase substrateL-arginine at selected concentrations. To examine the physiologicalrelevance of arginase inhibitors in IAS relaxation, the influence ofselected concentrating of arginase inhibitorg on EFS-induced IASrelaxation was examined.

At the end of each experiment, smooth muscle strips were treated with 5millimolar EDTA to establish the maximal relaxation (Biancani et al.,1985, Gastroenterology 89:867-874). Each smooth muscle strip served asits own control.

Tissue Preparation

Tissue samples of the opossum internal anal sphincter muscle (IAS),adjoining rectal tissue, liver, and brain were homogenized using anUltraturrax™ tissue homogenizer (Tekamr, Cincinnati, Ohio) in a solutioncomprising 10 millimolar Tris-HCl, 150 millimolar KCl, and 25 millimolarMnCl₂, at pH 7.4. The homnogenates were dialyzed overnight against thesame solution. Dialyzed homogenates were centrifuged to remove insolublematerial and concentrated with Amicon Centricon™ 30 microconcentratorsto yield stock protein preparations having protein concentrations of 2.4milligrams per milliliter for IAS smooth muscle, 3.6 milligrams permilliliter for rectal smooth muscle, 3 milligrams per milliliter forbrain, and 17.5 milligrams per milliliter for liver. Proteinconcentrations were estimated with the Pierce Coomassie Protein Reagentkit using bovine serum albumin as a standard.

Arginase Assay

Arginase activity in tissue homogenates was evaluated using theradioactive L-[guanidino¹⁴C]arginine assay of Rüegg and Russell (1980,Anal. Biochem. 102:206-212). Assays were performed using a solutioncomprising 100 millimolar CHES-NaOH and 0.1 millimolar MnCl₂ at pH 9.0,The reactions were initiated by addition of 5 microliters of tissuehomogenate to 45 microliters of reaction mixture that contained the CHESbuffer, the appropriate concentration of arginine (0.5 to 5 millimolar)and about 5.0×10⁴ counts per minute of L-[guanidino-¹⁴C]arginine. IAS,rectal muscle, and brain homogenates were incubated with the assaymixture at room temperature for one hour, and the liver sample wasincubated for 5 minutes. The reactions were stopped by addition of 200microliters of a stop solution containing 0.25 molar acetic acid, 7molar urea, and 10 millimolar arginase at pH 4.5. Arginase hasessentially no activity at the low pH of the stop solution. [¹⁴C] Ureawas separated from non-reacted L-[guanidino-¹⁴C]arginine by treatmentwith 200 μl of 1:1 v/v slurry of Dowex 50W-X8 in water, and quantitatedby adding 200 microliters of the supernatant from the Dowex treatment to3 milliliters of Liquiscint™ (National Diagnostics) for liquidscintillation counting in a Beckman LS 5000CE counter. The data wereanalyzed using double-reciprocal plots of the initial velocitymeasurements; standard errors were determined by regression analysis.

Arginase Inhibition Studies

Assays in the presence of the inhibitors N^(ω)-hydroxy-L-arginine (1 to100 micromolar) and ABHA (0.05 to 100 micromolar) were performed asdescribed above. The amino acid D-ornithine served as a control in theseexperiments, since this amino acid, unlike L-ornithine, is not aninhibitor of arginase.

Drugs and Chemicals

Bovine liver arginase, N^(ω)-hydroxy-L-arginine (L-HO-Arg),N^(ω)-nitro-L-arginine (L-NNA), N^(ω)-nitro-D-arginine methyl ester(D-NNA), L-arginine hydrochloride, D-arginine, D-ornithine and atropinesulfate were obtained from Sigma Chemical Co., St. Louis, Mo.Guanethidine monosulfate was from Ciba Pharmaceuticals (Summit, N.J.).Ethylenediamine tetraacetic acid (EDTA) tetrasodium salt was from FisherScientific Co., Fair Lawn, N.J. ABHA was synthesized as described inExample 2. L-[guanidino-¹⁴C]Arginine (specific activity 2.5 GBq permillimole) was from NEN/Dupont.

All chemicals used were of the highest purity available. Solutions ofall the chemicals were prepared in Krebs' solution on the day of thecorresponding experiment.

Data analysis

The responses to EFS and other relaxants were expressed as a percentageof maximal relaxation caused by 5 millimolar EDTA. The results areexpressed as means±standard error. Statistical significance betweendifferent groups were determined using t-test and a p value smaller than0.05 was considered significant.

The Results of the experiments presented in this Example are nowdescribed.

Influence of Exogenous Administration of Arginase Before and AfterArginase Inhibitors N^(ω)-hydroxy-L-arginine (L-HO-Arg) and2(S)-Amino-6-boronohexanoic Acid (ABHA) on NANC Nerve-mediatedRelaxation of IAS

First, the effects of selected concentrations of arginase on IASrelaxation by the NANC nerve stimulation were determined. About 30 unitsper milliliter arginase was found to be optimal for attenuating NANCnerve mediated relaxation of IAS. In the experiments used to examine theinfluence of L-HO-Arg, IAS relaxations in response to 0.5, 1 and 2 Hertzof EFS in control experiments were 31.0±0.7, 55.9±3.0 and 67.7±3.9%,respectively. Following arginase pre-treatment, IAS relaxation inresponse to EFS was significantly suppressed and these values inresponse to 0.5, 1 and 2 Hertz EFS were 12.1±2.5, 28.2±7.3 and36.6±7.4%, respectively (FIG. 18; p<0.05; n=5). Pre-treatment of tissueswith L-HO-Arg (1×10⁻⁴ molar) before addition of arginase antagonized theeffect of arginase in attenuating EFS-induced IAS relaxation.

The effect of ABHA in antagonizing the arginase-suppressed IASrelaxations was similar to that of L-HO-Arg (FIG. 19; p<0.05; n=5).

Influence of Arginase Inhibitors on NANC Nerve-Mediated IAS Relaxationin the Presence of the NO synthase Inhibitor L-NNA

It is known that the NO synthase inhibitor L-NNA causes a markedsuppression of IAS relaxation by NANC nerve stimulation. In order totest the selectivity of the arginase inhibitors in IAS, their effects onIAS relaxation suppressed by the NO synthase inhibitor L-NNA (3×10⁻⁶molar) were examined. The results obtained were compared with reversalof the NANC relaxation of IAS caused by the NO synthase substrateL-arginine at selected concentrations. Interestingly, L-NNA-suppressedIAS relaxation was completely reversed by L-HO-Arg (FIG. 20). In controlexperiments, the decrease in basal IAS tension in response to 0.5, 1, 2and 5 Hertz EFS was 26.7±3.6, 46.4±4.0, 64.1±3.0 and 74.8±3.9%respectively. L-NNA significant attenuated IAS relaxation to 0±0,0.6±0.6, 1.8±1.1 and 5.4±2.1%, respectively (p<0.05; n=5). NANCnerve-mediated IAS relaxation in the presence of both L-NNA and L-HO-Arg(3×10⁻⁴ molar) was indistinguishable from that of control values(p<0.05; n=5). In this regard, L-HO-Arg was nearly as potent asL-arginine in causing the reversal (FIG. 21).

Interestingly, and in contrast to L-HO-Arg, the newly synthesizedarginase inhibitor ABHA (3×10⁻⁴ molar) did not cause any reversal ofL-NNA-suppressed IAS relaxation (FIG. 22; p<0.05; n=4).

Influence of the Arginase Inhibitors on IAS Relaxation caused by NANCNerve Stimulation

In order to determine the physiological significance of arginase ingastrointestinal smooth muscle, the effects of arginase inhibitors onNANC nerve-mediated IAS relaxation were examined. Interestingly, bothL-HO-Arg (FIG. 23) and ABHA (FIG. 24) caused significant andconcentration-dependent augmentation of NANC nerve-mediated IASrelaxation by EFS. This was particularly evident at lower EFSfrequencies. In control experiments for these series of studies, thedecrease in IAS tension with 0.5 and 1 Hertz EFS before and afterL-HO-Arg (3×10⁻⁴ molar) was 35.5±7.0 and 54.7±7.0, and 57.4±4.9,68.8±4.9%, respectively (p<0.05; n=4). Similar data were obtained inexperiments involving ABHA: 29.3±5.7, 49.6±3.9 and 56.5±7.0, 73.6±3.5%,respectively of the decrease in basal IAS tension before and afteraddition of selective arginase inhibitor (1×10⁻⁴ molar; p<0.05; n=4).

Basal Levels of Arginase Activity in Different Tissues

A comparison of basal arginase activity in selected tissues is shown inFIG. 25. Among the tissues examined, liver tissue contained the highestlevels of arginase activity (7,400 nanomoles per minute per milligram ofprotein), consistent with the role of this tissue in nitrogen metabolismand urea synthesis. Among the non-hepatic tissues tested, IAS containedthe highest levels of arginase activity (7.8 nanomoles per minute permilligram of protein), while rectum and brain tissues exhibited lowerlevels (1.7 nanomoles per minute per milligram of protein). K_(m) valuesfor enzymes obtained from each of these tissues were similar, rangingfrom 1.0 to 1.9 millimolar. These K_(m) values are comparable to thosefor the native and recombinant rat liver enzymes (Cavaili et al., 1994,Biochemistry 33:10652-10657).

Influence of Arginase Inhibitors L-HO-Arg and ABHA on Basal ArginaseActivity in Selected Tissues

Among tissues investigated, L-HO-arg was the most potent inhibitorarginase activity in the liver (FIG. 26). IC50 values for the inhibitionof arginase activity by L-HO-arg in liver, IAS, rectum, and brainhomogenates were 2.4, 25, 42, and 40 micromolar, respectively. Thus,liver arginase activity was approximately 10-20-fold more sensitive toinhibition by L-HO-Arg than the arginase activities in the othertissues. The ability of ABRA to inhibit arginase activity in the tissueswas in striking contrast to the inhibition observed with L-HO-Arg. ABHAwas the most potent inhibitor of arginase activity in brain and rectumtissues, and also inhibited arginase in IAS and liver. The correspondingIC₅₀ values were 0.05 (brain), 0.05 (rectum), 0.10 (IAS), and 0.44(liver) micromolar (FIG. 27). Inhibition constants for ABHA wereestimated by titrating the inhibitor in assay mixtures containing aselected arginine concentration fixed at the K_(m) value, and assumingcompetitive inhibition. These experiments yielded estimated K_(i) valuesof 0.018, 0.026, 0.05, and 0.19 micromolar for arginase activities inbrain, rectum, IAS, and liver, respectively. The estimated K_(i) forABHA inhibition of the liver enzyme is in good agreement with the K_(d)of 0.11 micromolar determined by titration calorimetry.

Influence of the NO synthase Inhibitor L-N-Nitro-Arginase (L-NNA) andL-NNA Plus the Arginase Inhibitors on the arginase Activity in DifferentTissues

It is known that L-HO-Arg is not only an intermediate in thebiosynthesis of NO, but is also a substrate for NO synthase. Thus, it ispossible that tissue variations in inhibition of arginase activities byL-HO-Arg could be due to depletion of added L-HO-Arg by conversion toNO. In order to test this possibility, the effects of L-HO-Arg onarginase activities in the tissue homogenates were determined in thepresence of the NO synthase inhibitor L-NNA. L-NNA, at 80 micromolar,had no effect on the activities of the various arginases in the presenceof L-NO-Arg (FIG. 28), indicating that depletion of added L-HO-Arg bythe action of NO synthase was not a concern in these experiments.Furthermore, the results indicate that tissue-specific variations inarginase inhibition by L-HO-Arg are likely to result from inherentdifferences in the arginase enzymes expressed in these tissues.

These studies demonstrate that ABHA is a potent, tissue-selectiveinhibitor of arginase. ABHA was 5,250,840, and 800 times more potentthan L-HO-Arg in inhibiting the arginase activity in liver, IAS, rectum,and brain homogenates, respectively. Among the tissues examined, ABHAwas more potent in inhibiting brain, rectum, and IAS arginase activitythan the liver, with estimated K_(i) values of 0.018, 0.026, and 0.05micromolar, respectively, assuming competitive inhibition. Althoughcomplete inhibition patterns were not determined, previous studies haveshown that ABHA can displace the competitive inhibitor L-HO-Arg from therat liver enzyme (Example 2). In contrast to the inhibition results withABHA, L-HO-Arg was more potent in inhibiting arginase activity in liverhomogenates than in the other tissues (FIG. 26).

Two isozymes of arginase have been described in mammals, the hepatic(type I) arginase and non-hepatic type (type II) arginase (Jenkinson etal., 1996, Comp. Biochem. Physiol. 114B:107-132; Buga et al., 1996, Am.J. Physiol. Heart Circ. Physiol. 271: H1988-H1998; Daghigh et al., 1994,Biochem. Biophys. Res. Commun. 202:174-180; Boucher et al., 1994,Biochem. Biophys. Res. Commun. 203:1614-1621; Hecker et al., 1995, FEBSLett. 359:251-254; Gotoh et al., 1996, FEBS Lett. 395:119-122). 122).Type I arginase is found predominantly in mammalian liver and red bloodcells, while the type II enzyme is thought to be expressed inmacrophages, kidney and endothelial cells. Although the expressionpatterns for the arginase in opossum rectum, brain, IAS, and liver arenot known, the differential effects of ABHA and L-HO-Arg on arginaseactivities in these tissues are consistent with type II enzymeexpression in the non-hepatic tissues.

The higher potency of L-HO-Arg against hepatic arginase, as compared tothe non-hepatic tissue extracts might be related to the ability ofL-HO-Arg to serve as a substrate for NO synthase. Thus, in tissuesexpressing high levels of NO synthase, L-HO-Arg would be rapidlyconverted to NO and citrulline, lowering the effective concentration ofL-HO-Arg and decreasing arginase inhibition. To assess this possibility,inhibition studies with L-HO-Arg were repeated in the presence of L-NNA,a known inhibitor of NO synthase. Control experiments established thatL-NNA had no effect on the arginase activities of the various tissues.The combination of L-NNA and L-HO-Arg was no more effective thanL-HO-Arg alone for inhibiting arginase activities, indicating that thedifferential inhibition of the arginase activities in liver, tow brain,rectum, and IAS is not attributable to NO synthase depletion ofL-HO-Arg, and is therefore likely to reflect differences in affinity ofthe arginase for the inhibitor.

The functional data in IAS also indicate that ABHA is a more selectiveinhibitor of arginase than is L-HO-Arg. This was evident from IASstudies in the presence of the NO synthase inhibitor L-NNA. L-HO-Arg canserve as an NO synthase substrate. The experiments described in thisExample were performed to examine the influence of L-HO-Arg on NANCrelaxation of IAS that was attenuated by the NO synthase inhibitor. Theeffects of L-HO-Arg and ABHA in reversing attenuation of NANC relaxationwere compared with L-arginine, an authentic substrate for NO synthase.Interestingly, L-HO-Arg reversed L-NNA-attenuated NANC relaxation of IASwith a potency comparable to L-arginine. ABHA, on the other hand, had noeffect on IAS relaxation suppressed by the NO synthase inhibitor. Thissuggests that L-HO-Arg may in part be a substrate for NO synthase andthat it may be a less selective inhibitor of arginase than is ABHA.

Before the present study, there had been only limited information on thephysiological relevance of arginase in NANC nerve-mediated relaxation ingastrointestinal smooth muscle. The data in this Example demonstratethat arginase inhibitors L-HO-Arg and ABHA augment NANC nerve-mediatedrelaxation of the IAS. Because NO synthase pathway is the predominantpathway responsible for the NANC nerve-mediated relaxation of IAS(Rattan and Chakder, 1992, Am. J. Physiol. Gastrointest. Liver Physiol.262:G107-G112; Rattan et al., 1992, Gastroenterology 103:43-50),augmentation of IAS relaxation is believed to be due to up-regulation ofthe NO synthase pathway induced by an increase in tissue levels ofL-arginine.

It has been established that exogenously administered L-arginine has nosignificant effect on NANC relaxation of IAS unless the tissues areL-arginine deficient (Chakder and Rattan, 1997, J. Pharmacol. Exp. Ther.282:378-384). In the basal state in normal tissues, exogenous L-argininehas no significant effect on either basal IAS tone or NANC relaxation(Chakder and Rattan, 1997, J. Pharmacol. Exp. Ther. 282;378-384).Conversely, in L-arginine-deficient tissues, exogenous L-arginine causesa significant decrease in basal IAS tone and reversal of impaired NANCrelaxation in IAS smooth muscle (Chakder and Rattan, 1997, J. Pharmacol.Exp. Ther. 282:378-384; Rattan and Chakder, 1997, Gastroenterology112:1250-1259).

It is believed that the effect of exogenous L-arginine in L-argininedeficient tissues is attributable to an increase in arginine uptakewhich leads to augmentation of NANC relaxation via up-regulation of theNO synthase pathway. Such mechanisms may not be operative in normaltissues due to the normal state of equilibrium of L-arginine levels atcellular levels. Augmentation of NANC relaxation in the presence ofarginase inhibitors can be due to an increase in intracellularL-arginine levels, as proposed in other non-gastrointestinal tissues(Jenkinson et al., 1996, Comp. Biochem. Physiol. 114b:107-132; Buga etal., 1996, Am. J. Physiol. Heart Circ. Physiol. 271:H1988-H1998; Boucheret al., 1994, Biochem. Biophys. Res. Commun. 203:1614-1621; Hecker etal., 1995, FEBS Lett. 359:251-254).

In IAS, ABHA was about 250 times more potent an arginase inhibitor thanL-HO-Arg. However, in smooth muscle NANC relaxation experiments, thesetwo agents were approximately equipotent for augmenting NANC relaxation.In the functional data, smooth muscle relaxation is the final outcome ofmultiple pathways that involve not only arginase but also NO synthase.Net IAS smooth muscle relaxation in response to NANC nerve stimulationin the presence of inhibitors is the result of their interaction withdifferent pathways available to them. L-HO-Arg acts as both an arginaseinhibitor and a substrate for NO synthase. ABHA, on the other hand, isselective for arginase inhibition only (Rattan and Chakder, 1992, Am. J.Physiol. Gastrointest. Liver Physiol. 262:G107-G112; Rattan et al.,1992, Gastroenterology 103:43-50; Chakder and Rattan, 1993, Am. J.Physiol. Gastrointest. Liver Physiol. 264:G7-G12. Others have shown thatthe NO synthase pathway is the predominant pathway for NANCnerve-mediated smooth muscle relaxation(Tottruo et al, 1992,Gastroenterology 102:409-415; O'Kelly et al., 1993, Gut 34:689-693).Therefore, augmentation of NANC relaxation in IAS by L-HO-Arg can be dueto the summation of its effects.

An increase in tissue arginase levels has been associated with a numberof pathological conditions, including gastric cancer (Wu et al., 1992,Life Sci. 51:1355-1361; Leu and Wang, 1992, Cancer 70:733-736; Straus etal., 1992, Clin. Chim. Acta 210:5-12; Ikemoto et al., 1993, Clin. Chem.39:794-799; Wu et al, 1994, Dig. Dis. Sci 39:1107-1112). Additionally,elevated arginase levels following human orthotopic livertransplantation have been shown to cause pulmonary hypertension andreduced hepatic blood flow (Langle et al., 1997, Transplantation63:1225-1233; Langle et al., 1995, Transplantation 59:1542-1549). Higherblood levels of arginase have been found in patients with various tumors(Wu et al., 1992, Life Sci. 51:1355-1361; Leu and Wang, 1992, Cancer70:733-736; Straus et al., 1992, Clin. Chim. Acta 210:5-12; Wu et al.1994, Dig. Dis. Sci., 39:1107-1112; Paranuli and Singh, 1996, CancerLett., 107:249-256) and in patients afflicted with certain forms ofhepatic injury (Ikemoto et al., 1993, Clin. Chem. 39:794-799). Arginaseinhibitors therefore can have a significant role in pathophysiology andpotential therapy in a number of disorders in humans and other mammals.

In addition to its therapeutic potential, ABHA can have a novel role inthe identification of isozyme-specific arginase pathways and their roleas therapeutic potential for the specific control of the hemodynamiceffects associated with the unregulated arginage activity.

EXAMPLE 5 Arginase-Boronic Acid Complex Highlights a Physiological Rolein Erectile Function

The crystal structure of the complex formed between the bi-nuclearmanganese metalloenzyme arginase and 2(S)-amino-6-boronohexanoic acid(AB HA) has been determined at 1.7 angstrom resolution from a crystalperfectly twinned by hemihedry. ABHA binds as the tetrahedral boronateanion, with one hydroxyl oxygen symmetrically bridging the bi-nuclearmanganese cluster and a second hydroxyl oxygen coordinating to Mn²⁺_(A). This binding mode mimics the transition state of a metal-activatedhydroxide mechanism. This transition state structure differs from thatoccurring in NO biosynthesis, thereby explaining why AHA does notinhibit NO synthase. Arginase activity is present in the penis. ABHAcauses significant enhancement of non-adrenergic, non-cholinergic (NANC0nerve-mediated relaxation of penile corpus cavenosum smooth muscle,indicating that arginase inhibition sustains L-arginine concentration ata sufficiently high level in the muscle that NO synthase is active.Thus, the experiments presented in this Example demonstrate that humanpenile arginase is a target for therapeutic intervention in treatment oferectile dysfunction.

The materials and methods used in the experiments presented in thisExample are now described.

Crstallization and Data Collection

Crystals of arginase-ABHA complex were prepared at room temperature(i.e. about 20° C.) by equilibrating a hanging drop containing 5microliters of protein solution (11 milligrams per milliliter ofarginase, 2 millimolar ABHA, 4 millimolar MnCl₂, 2 millimolarbeta-mercaptoethanol, and 25 millimolar bicine {pH 8.5}, and 5microliters of precipitant solution {26% polyethylene glycol 1500, 100millimolar bicine <pH 8.1>}) against 1 milliliter of precipitantsolution in the well reservoir. hexagonal rod-shaped crystals withapproximate dimensions of 0.1×0.1×0.5 millimeters appeared within 2weeks. Diffraction data were collected from a single flash-cooledcrystal of the arginase-ABHA complex at CHESS (Cornell High EnergySynchotron Source) beamline A-1, and intensity data integration andreduction were performed using DENZO and SCALEPACK software,respectively, as described (Otwinowski et al., 1997, Meth. Enzymol.276:307-326).

Phasing and Refinement of the Twinned Structure

Initial phasing by molecular replacement with AMoRe software (Navaza,1994, Acta Crystallogr. A50:157-163) was achieved using the structure ofthe native rat liver arginase monomer (Kanyo et al., 1996, Nature383:554-557) as a search probe. Using intensity data in the 20-3angstrom shell with 1>3σ, the cross rotation search yielded twoequivalent 10.7σ peaks at α=34.9°, β=126.4°, γ=283.0° and α=94.9°,β=126.6°, γ=283.3° (next highest peak=5.6σ). Subsequent translationsearches yielded 13.9σ and 10.8σ peaks corresponding to fractionalcoordinates x=0.2436, y=0.2756, z=0.0000 and x=0.3330, y=0.9348,z=0.5009 (next highest peak=2.6σ). Rigid body refinement of thissolution lowered the crystallographic R factor from 0.503 to 0.379.Monomer positions in twin domain B were generated by applying the twinoperation to the molecular replacement model.

Iterative rounds of refinement and rebuilding of the native model wereperformed using CNS and O, as described (Brünger et al., 1998, ActaCrystallogr. D49:375-380; Jones et al., 1991, Acta Crystallogr.A47:110-119). For refinement with CNS against the measured twinnedstructure factor amplitudes |F_(obs)|, the target residual was based onthe numerator of R_(twin) (Table 8) and implemented in CNS version 0.5.Each twin domain was defined as an ‘alternate conformation’ withoccupancy set to ½, and interactions between mutually exclusive twindomains were disengaged.

After each round of CNS refinement, the in-progress atomic model wasused to deconvolute each measured intensity I_(obs) into estimatedcrystallographic intensities I_(obs/A) and I_(obs/B) corresponding totwin domains A and B. For omit map calculation, residues were deletedprior to the structure-based deconvolution of I_(obs) in order tominimize model bias. A bulk solvent term was included in the model priorto calculation of structure factor amplitudes, which improved thequality of the electron density maps; map averaging across both twindomains additionally improved map quality. In the final stages ofrefinement, the inhibitor ABHA was built into clear and unbiasedelectron density when R_(twin) decreased to 0.184. Strictnon-crystallographic symmetry constraints were employed duringrefinement, and these were relaxed to appropriately weighted restraintsas judged by R_(twin/free). Data collection and refinement statisticsare reported in Table 8.

Organ Bath Experiments Penile cavernosal tissue strips were obtainedfrom male New Zealand white rabbits (3.0-3.5 kilogram body weight) andmounted in organ bath preparations, as described (Kim et al., 1991, J.Clin. Invest. 88:112-118). Tissues strips at optimal isometric tensionwere contracted using 40 nanomolar endothelin-1 and subjected toelectrical field stimulation (EFS), by means of two platinum plateelectrodes positioned on either side of the tissue and a currentamplifier in series with a square pulse stimulator. Each stimulationperiod lasted 20 seconds with trains of square waves having a pulseduration of 0.5 milliseconds and a potential difference of 10 volts.Frequency was varied from 1 to 10 Hertz. For all experiments, NANCresponses were isolated in tissue strips by treatment with indomethacinto inhibit vasoactive prostanoid synthesis, 10 micromolar bretylium toinhibit adrenergic neurotransmission, and 1 micromolar atropine to blockmuscarinic acetylcholine receptors. All tissues were first subject toEFS in the absence of ABHA, and then stimulations were repeated in thepresence of increasing concentrations (0.1 to 1.0 millimolar) ABHAfollowing a 20 minute incubation period. At the end of each experiment,all tissue strips were treated with 10 micromolar papaverine and tomicromolar nitroprusside to induce maximal relaxation (100%). For eachexperiment, the response at each frequency in the presence of ABHA wascompared to control responses using a paired t-test method. Responseswere further analyzed by determining the ratios of the responses in thepresence and absence of ABHA treatment. Comparisons were judgedstatistically significant if the two-tailed p-value≦0.05.

Nitric Oxide Synthase-ABHA Assay

NADPH, FAD, FMN, L-arginine,N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid (HEPES)(6R)-5,6,7,8-tetrahydro-L-biopterin (BH₄), aminoguanidine, CaCl₂, andphosphodiesterase cyclic nucleotide activator (calmodulin) werepurchased from Sigma Chemical Company (St. Louis, Mo.). Neuronal NOsynthase was purchased from Biomol, dialyzed to removebeta-mercaptoethanol, and concentrated to 0.1 milligram per milliliter.The nitric oxide synthase colorimetric assay kit was purchased fromCalbiochem. Materials were used without further purification. The effectof ABHA on NO synthesis was assayed by monitoring the sum of NO₂ ⁻ andNO₃ ⁻ production, as described (Verdon et al., 1995, Anal. Biochem.224:502-508). The enzyme solution was shaken at 37° C. for 15 minuteswith 50 millimolar HEPES (pH 7.5), 100 micromolar NADPH, 4 micromolarFAD, 4 micromolar FMN, 6 micromolar BH₄, 100 micromolar L-arginine,0.0-1.0 millimolar ABHA, and 0.02 milligram per milliliter NO synthasein a final volume of 100 microliters. For experiments in which neuronalNO synthase was used, 1 millimolar CaCl₂ and 100 micrograms permilliliter calmodulin were also added. Reactions were stopped byincubating the reaction mixture at 100° C. for 3 minutes. Nitrate wasreduced to nitrite using nitrate reductase (40 minutes at roomtemperature), and the remaining NADPH was depleted by incubation withlactate dehydrogenase and sodium pyruvate for 20 minutes at roomtemperature. Addition of the Greiss reagents, sulfanilamide andN-(1-naphthyl)-ethylenenediamine, converted all available nitrite into adeep purple azo compound. Absorbance at 540 nanometers was used toquantify the extent of NO_(x) production.

Measurement of Arginase Activity

Human penile cavernosal tissue was obtained from patients undergoingimplantation of penile prostheses, as described (Kim et al., 1991, J.Clin. Invest. 88:112-118). Human and rabbit cavernosal tissue was frozenin liquid nitrogen and pulverized. The resulting tissue powder wascombined 1:4 (weight:volume) with ice-cold buffer comprising 20millimolar HEPES (pH 7.4), and 0.25 molar sucrose. The mixture washomogenized on ice in the presence of protease inhibitors (PMSF,leupeptin, and aprotinin at protease-inhibiting concentrations) using aBrinkmann PT3000 polytron. The homogenate was centrifuged at 3000×g for20 minutes, and the supernatant was used for enzyme assay. Arginaseactivity was assayed as described (Rüegg et al., 1980, Anal. Biochem.102:206-212). Briefly, 10 microliter aliquots of tissue extract wereincubated with increasing concentrations of non-labeled L-arginine and100,000 dpm of [14C-guanidino]-L-arginine (obtained from NEN LifeScience Products) in 90 microliters of buffer (74 millimolar glycine{pH9.7}, 0.25 millimolar MnCl₂) for 60 minutes at 25° C. To terminatethe reaction, 400 microliters of 0.25 molar acetic acid (pH 4.5), 7molar urea, and 10 millimolar L-arginine was added to each tube. Afteraddition of 500 microliters of water, samples were passed through a0.5-milliliter column of Dowex™ 50W-X8 resin (obtained from Bio-RadLaboratories). Tubes were rinsed twice with 500 microliter aliquots ofwater, and both rinses were poured onto the column. Finally, columnswere washed with 1 milliliter of water. All effluent was collected in 20milliliter vials and combined with 16 milliliters of Liquiscint™(obtained from National Diagnostics). Radioactivity was quantified byliquid scintillation spectroscopy using a Packard Tri-Carb™ 2300TRAnalyzer. All measurements were performed in 6 replicate samples.

Crystallographic Coordinates

Coordinates of the arginase-ABHA complex were deposited in the ProteinData Bank, and were assigned accession code 1D3V.

The results of the experiments presented in this Example are nowdescribed.

Structure Determination from a Twinned Arginase Crystal

A single crystal of the arginase-ABHA complex diffracted to 1.7 angstromresolution at CHESS. The data collection and refinement statistics forthis crystal are listed in Table 8.

TABLE 8 Resolution (Angstroms) 1.7 Total reflections 126,870 Uniquereflections 66,822 Completeness 91.7% R_(merge) ¹ 0.053 Reflections usedin refinement (>2σ) 64,734 Protein atoms (N)² 2,345 Inhibitor atoms (N)²13 Solvent atoms (N)² 150 Manganese ions (N)² 2 R_(twin) ³ 0.157R_(twin/free) ³ 0.179 R.m.s. deviations: Bonds (angstroms) 0.008 Angles(degrees) 1.4 Dihedrals (degrees) 23.1 Impropers (degrees) 0.9¹R_(merge) = Σ|I_(i) − <I_(t)>|/Σ|<I_(i)>|, where I_(i) is the intensitymeasurement for reflection i, and <I_(i)> is the mean intensitycalculated for reflection i from replicate data. ²per monomer ³R_(twin)= Σ(|F_(obs)| − [|F_(calc/A)|² + |F_(calc/B)|²]^(½))/Σ|F_(obs)|, where|F_(obs)| is observed structure factor amplitude derived from twinnedintensity I_(obs), and |F_(calc/A)| and |F_(calc/B)| are structuralfactor amplitudes calculated for the separate twin domains A and #B,respectively. R_(twin) underestimates the residual error in the model byaveraging the difference between observed and calculated structurefactor amplitudes over the two twin-related reflections. The sameexpression describes R_(twin/free), which was calculated for 3,331 testset reflections held aside during refinement.

Diffraction intensities exhibited symmetry consistent with space groupP6 (unit cell parameters a=b=91,3 angstroms, c=69.6 angstroms; onemonomer in the asymmetric unit), but this assignment was inconsistentwit the molecular symmetry of the arginase trimer. Subsequent analysisof measured intensities revealed deviations from Wilson statistics with<I²>/<I>²=about 1.5 for thin resolution shells, indicative of perfecthemihedral twinning that obscured the true crystallographic symmetry ofspace group P3. Two monomers (from two separate trimers) occupy theasymmetric unit.

Initial phasing was achieved by molecular replacement. Ultimately, fourindependent copies of the arginase monomer were accounted for—the two inthe asymmetric unit and their two twins. For the calculation of electrondensity maps, structure factor amplitudes were calculated fromdeconvoluted intensities using the structure-based algorithm of Redinboet al (1993, Acta Crystallogr. D49:375-380). The structure of theenzyme-inhibitor complex was refined against |F_(obs)| simultaneously inboth twin domains using CNS with a modified target residual, asdescribed (Brünger et al., 1998, Acta Crystallogr. D54:905-921).

Mechanistic Inferences from Inhibitor Binding

The trigonal planar boronic acid group of ABHA is hydrated to form thetetrahedral boronate anion in the arginase-ABHA complex, as illustratedin FIGS. 29A and 29B. A single boronate hydroxyl group (O1)symmetrically bridges the bi-nuclear manganese cluster (Mn²⁺ _(A)—O andMn²⁺ _(B)—O separations=2.2 angstroms) and also donates a hydrogen bondto Oδ2 of Asp-128. This binding mode mimics the first step of theproposed arginase mechanism (see, e.g. Kanyo et al., 1996, Nature383:554-557), in which a metal-bridging hydroxide ion attacks thetrigonal planar guanidinium group of L-arginine to form a tetrahedralintermediate bridging both metal ions, as illustrated in FIG. 29C.

A second boronate hydroxyl group (O2) coordinates to Mn²⁺ _(A) with alonger Mn—O separation of 2.4 angstroms. Therefore, inhibitor bindingchanges the geometry of the Mn²⁺ _(A) coordination polyhedron fromsquare pyramidal in the native enzyme to distorted octahedral in theenzyme-inhibitor complex. There is no net change in the coordinationgeometry of Mn²⁺ _(B), which remains distorted octahedral. The Mn²⁺_(A)—Mn²⁺ _(B) separation increases slightly, from 3.3 angstroms in thenative enzyme to 3.4 angstroms in the enzyme-inhibitor complex.

The negatively charged carboxylate group of Glu-277 does not hydrogenbond to both boronate hydroxyl groups O2 and O3 (O—O separations=3.4angstroms and 3.8 angstroms), contrary to the expectations of Baggio etal. (1997, J. Amer. Chem. Soc. 119:8107-8108). However, modelingexperiments indicate that a double salt link between Glu-277 and thesubstrate would place the scissile guanidinium group directly overmetal-bridging hydroxide ion (Kanyo et al., 1996, Nature 383:554-557).Additionally, L-arginine hydrogen bonds with this glutamate residue inthe inactivated arginase from B. caldovelox arginase (Bewley et al.,1999, Structure 7:435-448). The lack of a corresponding hydrogen bond inthe arginase-ABHA complex may result from electrostatic repulsion withthe negatively charged boronate anion. Such a repulsive interactionwould not occur with the neutral tetrahedral intermediate occurring inthe arginase mechanism (see FIG. 29C).

An extensive network of hydrogen bond interactions with the alpha-aminoand alpha-carboxylate groups of ABHA reveal the structural andstereochemical basis for substrate specificity toward free L-arginine,as illustrated in FIG. 29B (see also Reczkowski et al., 1994 Arch.Biochem. Biophys. 312:31-37). Alteration of the alpha-amino oralpha-carboxylate groups of L-arginine would significantly compromiseenzyme-substrate hydrogen boding, recognition, and catalysis. Notably,most of these hydrogen bond interactions are conserved in the complex ofL-arginine with the inactivated arginase from B. caldovelox.

Arginase and the Physiology of Penile Erection

The arginase inhibitor ABHA is the most potent and most stable inhibitorof arginase reported to date. Because the tetrahedral intermediate inthe NO synthase mechanism (see Stuehr et al., 1992, Adv. Enzymol.65:287-346) cannot be mimicked by the ABHA boronate anion, ABHA does notinhibit NO synthase (e.g. no inhibition of NO synthase is observed,either at a concentration 0.1 millimolar or 1.0 millimolar ABHA). Withthe discovery, described herein, that arginase activity occurs in corpuscavenosum tissue extracts prepared from rabbit penis and human penis, itis now possible to probe the physiological relationship between arginaseand NO synthase in penile corpus cavenosum.

The effects of ABHA on smooth muscle contractility was studied usingorgan bath preparations of rabbit penile corpus cavenosum. Tissue stripswere procured and prepared, and NANC nerve-mediated responses wereelicited by EFS at varying frequencies. These responses are sensitive tothe neurotoxin tetrodotoxin, and are not dependent on the presence ofthe endothelium (Kim et al., 1991, J. Clin. Invest. 88:112-118; Saenz deTejada et al., 1988, Amer. J. Physiol. 254:H459-H467). Electricalstimulation caused frequency-dependent relaxation. Addition of ABHA inthe absence of electrical stimulation caused moderate relaxation due tobasal activity of NO synthase, as shown in FIGS. 30A and 30B. ABHAsignificantly enhanced relaxation in response to electrical stimulationin a dose-dependent manner, as illustrated in FIG. 30C. Thispotentiation was more noticeable at lower frequencies (i.e. not greaterthan about 2 Hertz). These results indicate that arginase can modulatethe production and availability of NO in penile corpus cavenosum.

Experiments described above using IAS muscle of opossum identifyarginase as the specific receptor for ABHA. This enzyme is responsiblefor enhanced relaxation. Specifically, arginase causes attenuation ofsmooth muscle relaxation by NANC nerve stimulation, and such relaxationcan be restored by addition of ABHA to tissue baths. In contrast, ABHAdoes not cause reversal of smooth muscle relaxation in the presence ofan NO synthase inhibitor such as N^(ω)-nitro-L-arginine. Taken together,these results indicate that arginase modulates production andavailability of NO in gastrointestinal smooth muscle, and in penilecorpus cavenosum.

Bioavailability of substrate L-arginine for NO biosynthesis can be afunction of dietary intake. For instance, dietary supplementation withL-arginine in an animal model resulted in increased levels of NOsynthase activity and enhanced erectile function without changing NOsynthase expression, suggesting that L-arginine concentrations in thepenis can be a substrate-limiting factor for NO synthase activity (seeMoody et al., 1997, J. Urol. 158:942-947). Furthermore, long-term oraladministration of L-arginine in patients with interstitial cystitisincreased NO-related enzymes and metabolites (Wheeler et al., 1997, J.Urol. 158:2045-2050). Oral administration of 2,800 milligrams per dayL-arginine improved erections in 40% of impotent but otherwise healthypatients in a pilot study (Zorgniotti et al., 1994, J. Impotence Res.6:33-35).

Although the connection between L-arginine bioavailability and NObiosynthesis is complex, enhancement of NANC nerve-mediated smoothmuscle tone effected by ABHA indicates that arginase has a role inmodulating L-arginine bioavailability for NO biosynthesis in the penis.These experiments demonstrate that the activity of human penile arginasecan be inhibited using ABHA or another arginase inhibitor describedherein in order to alleviate or inhibit erectile dysfunction.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention can be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed is:
 1. A method of inhibiting the activity of anarginase, the method comprising contacting the arginase with a compoundhaving the structure HOOC—CH(NH₂)—X¹—X²—X³—X⁴—B(OH)₂ wherein each of X¹,X², X³, and X⁴ is selected from the group consisting of —(CH₂)—, —S—,—O—, —(NM)—, and —(N-alkyl)—.
 2. The method of claim 1, wherein thearginase is yeast arginase.
 3. The method of claim 1, wherein thecompound hag a structure selected from the group consisting ofHOOC—CH(NH₂)—CH₂—CH₂—CH₂—CH—B(OH)₂ and HOOC—CH(N₂)—CH₂—S—CH₂—CH₂—B(OH)₂.4. The method of claim 3, wherein the compound is2(S)-amino-6-boronohexanoic acid.
 5. The method of claim 1, wherein thearginase is a mammalian arginase.
 6. The method of claim 5, wherein thearginase is a human arginase.
 7. The method of claim 6, wherein thearginase is a human type II arginase.
 8. A method of inhibiting theactivity of an arginase in a mammal, the method comprising administeringto the mammal a composition comprising a pharmaceutically acceptablecarrier and an arginase inhibitor having the structureHOOC—CH(NH₂)—X¹—X²—X³—X⁴—B(OH)₂ wherein each of X¹, X², X³, and X⁴ isselected from the group consisting of —(CH₂)—, —S—, —O—, —(NH)—, and—(N-alkyl)—.
 9. The method of claim 8, wherein the compound has astructure selected from the group consisting ofHOOC—CH(NH₂)—CH₂—CH₂—CH₂—CH—B(OH)₂ and HOOC—CH(NH₂)—CH₂—S—CH₂—CH—B(OH)₂.10. The method of claim 8, wherein the mammal is a human.
 11. The methodof claim 10, wherein the human comprises a tissue which exhibits anabnormally high level of arginase activity.
 12. The method of claim 10,wherein the human comprises a tissue which exhibits an abnormally lowlevel of nitric oxide synthase activity.
 13. A method of treating adisorder associated with an abnormally low level of nitric oxidesynthase activity in a tissue of a human, the method comprisingadministering to the human a composition comprising a pharmaceuticallyacceptable carrier and an arginase inhibitor having the structureHOOC—CH(NH₂)—X¹—X²—X³—X⁴—B(OH)₂ wherein each of X¹, X², X³, and X⁴ isselected from the group consisting of —(CH₂)—, —S—, —O—, —(NH)—, and—(N-alkyl)—.
 14. The method of claim 13, wherein the inhibitor has astructure selected from the group consisting ofHOOC—CH(NH₂)—CH₂CH₂—CH₂—CH—B(OH)₂ and HOOC—CH(NH₂)—CH₂—S—CH₂—CH₂—B(OH)₂.15. The method of claim 13, wherein the disorder is selected from thegroup consisting of heart disease, systemic hypertension, pulmonaryhypertension, erectile dysfunction, autoimmune encephalomyelitis,chronic renal failure, a gastrointestinal motility disorder, a gastriccancer, reduced hepatic blood flow, and cerebral vasospasm.
 16. A methodof relaxing smooth muscle in a mammal, the method comprisingadministering to the mammal a composition comprising a pharmaceuticallyacceptable carrier and an arginase inhibitor having the structureHOOC—CH(NH₂)—X¹—X²—X³—X⁴—B(OH)₂ wherein each of X¹, X², X³, and X⁴ isselected from the group consisting of —(CH₂)—, —S—, —O—, —(NH)—, and—(N-alkyl)—.
 17. The method of claim 16, wherein the inhibitor has astructure selected from the group consisting ofHOOC—CH(NH₂)—CH₂—CH₂—CH₂—CH₂—B(OH)₂ andHOOC—CH(NH₂)—CH₂—S—CH₂—CH₂—B(OH)_(2.)
 18. The method of claim 16,wherein the smooth muscle is selected from the group consisting ofgastrointestinal smooth muscle, anal sphincter smooth muscle, esophagealsphincter muscle, corpus cavernosum, sphincter of Oddi, arterial smoothmuscle, heart smooth muscle, pulmonary smooth muscle, kidney smoothmuscle, uterine smooth muscle, vaginal smooth muscle, cervical smoothmuscle, placental smooth muscle, and ocular smooth muscle.
 19. A methodof making a compound having the structureHOOC—CH(NH₂)—CH₂—CH₂—CH₂—CH₂—B,(OH)₂, the method comprising contacting amolecule of the tert-butyl ester of2(S)-N-(tert-butyloxycarbonyl)-6-[(1S,2S,3R,5S)-(+)-pinanedioxaboranyl]-hexanoicacid in an organic solvent with BCl₃.
 20. The method of claim 19,wherein the organic solvent is CH₂Cl₂.
 21. A method of making a compoundhaving the structure HOOC—CH(NH₂)—CH₂—CH₂—CH₂—CH₂—B(OH)₂, the methodcomprising the steps of (a) mixing a solution of the tert-butyl ester of2(S)-N-(tert-butyloxycarbonyl)-glutamic acid in tetrahydrofuran withtriethylamine and ethyl chloroformate to produce a first mixture,removing the resulting triethylammonium hydrochloride salt byfiltration, and treating the remaining mixture with an aqueous solutionof sodium borohydride to provide a first compound, wherein the firstcompound is the tert-butyl ester of2(S)-N-(tert-butyloxycarbonyl)-5-hydroxypentanoic acid; (b) subjectingthe first compound to Swem oxidation to produce a second compound; (c)subjecting the second compound to a Wittig reaction in the presence oftriphenylphosphonium methylide to produce a third compound; (d) mixing asolution of BH₃ with the third compound in the presence oftetrahydrofuran to produce a second mixture; (e) adding (1S,2S,3R,5S)-(+)-pinanediol to the second mixture to produce a fourthcompound, wherein the fourth compound is the tert-butyl ester of2(S)-N-(tert-butyloxycarbonyl)-6-[(1S,2S,3R,5S)-(+)-pinanedioxaboranyl]-hexanoicacid; and (f) mixing the fourth compound with BCl₃ in the presence ofCH₂Cl₂ to produce a compound having the structureHOOC—CH(NH₂)—CH₂—CH₂—CH₂—CH₂—B(OH)₂.
 22. A method of alleviatingerectile dysfunction in a human, the method comprising administering tothe human a pharmaceutical composition comprising an arginase inhibitorhaving the structure HOOC—CH(NH₂)—X¹—X²—X³—X⁴—B(OH)₂ wherein each of X¹,X², X³, and X⁴ is selected from the group consisting of —(CH₂)—, —S—,—O—, —(NH)—, and —(N-alkyl)—.
 23. The method of claim 22, wherein theinhibitor is 2(S)-amino-6-boronohexanoic acid.
 24. A method of treatinga disorder associated with an abnormally high level of arginase activityin a tissue of a human, the method comprising administering to the humana composition comprising a pharmaceutically acceptable carrier and anarginase inhibitor having the structure HOOC—CH(NH₂)—X¹—X²—X³—X⁴—B(OH)₂wherein each of X¹, X², X³, and X⁴ is selected from the group consistingof —(CH₂)—, —S—,—O—, —(NH)—, and —(N-alkyl)—.
 25. The method of claim24, wherein the inhibitor has a structure selected from the groupconsisting of HOOC—CH(NH₂)—CH₂—CH₂—CH₂—CH₂—B(OH)₂ andHOOC—CH(NH₂)—CH₂—S—CH₂—CH₂—B(OH)₂.
 26. The method of claim 21, whereinthe disorder is selected from the group consisting of heart disease,systemic hypertension, pulmonary hypertension, erectile dysfunction,autoimmune encephalomyelitis, chronic renal failure, a gastrointestinalmotility disorder, a gastric cancer, reduced hepatic blood flow, andcerebral vasospasm.